Agents for treatment of hcv and methods of use

ABSTRACT

An amphipathic helix at the approximate N-terminus of Hepatitis C virus (HCV) nonstructural proteins mediates the association of these proteins with cytoplasmic membranes in infected cells. This association is essential for replication. Thus, assessing the ability of compounds or protocols to disrupt the association of such helices with cytoplasmic membranes permits identification of compounds and protocols which are useful in the treatment of HCV infection. Also useful in the invention are mimics, or function-disrupting ligands, of an amphipathic helix of the nonstructural proteins described herein and antibodies and fragments thereof immunoreactive with said helix.

FIELD OF THE INVENTION

[0001] The invention relates generally to Hepatitis C virus (HCV)infection, and more specifically to interrupting the mechanism ofinfection by HCV and to methods to identify agents, which effect thisinterruption. The invention also relates to interfering with the abilityof HCV components to bind with cellular membranes of an infected cell.

BACKGROUND

[0002] Hepatitis C virus (HCV) establishes a chronic infection in a highpercentage of infected individuals and is associated with progressiveliver pathology, including cirrhosis and hepatocellular carcinoma.Antiviral drugs such as interferon alpha and ribavarin have had limitedsuccess in controlling HCV infection. As a result, it has become theleading cause for liver transplantation in the US. The HCV polyproteincomprises, from the amino terminus to the carboxy terminus, the coreprotein (C), the envelope proteins (E1 and E2), p7, a membrane boundprotein, whose function is unknown and the non-structural proteins (NS2,NS3, NS4A, NS4B, NS5A and NS5B) which are believed to be important forreplication. C codes for the core nucleocapsid protein, E1 and E2 areenvelope proteins that coat the virus, NS2, NS3 and NS4A are involved inproteolytic processing of the HCV polyprotein, and NS5B has RNApolymerase activity. The functions of NS4B and NS5A are unknown.

[0003] Hepatitis C virus is a significant cause of morbidity andmortality, infecting over 100,000,000 people worldwide. Annual HCVrelated costs in the United States are about $1 billion. Currenttherapies are clearly inadequate; the best available treatment atpresent is the combination of interferon and ribavirin, a treatmentwhich is inconveniently lengthy as it typically lasts over one and ahalf years, difficult to tolerate in that most patients have flu-likesymptoms, and extremely expensive as the cost is in the range ofthousands of dollars annually. Not only does the present treatment havethese disadvantages, but it is also not particularly effective.

[0004] Certain interactions of viral proteins with cell membranes havepreviously been described. For example, in poliovirus and Hepatitis Avirus, the nonstructural mammalian cells, or preferably using adenoviralor retroviral or other suitable viral vectors.

[0005] As described above, however, these competitor peptides need notbe the native sequences per se and need not even be peptides per se, butmay contain isosteric linkages or other polymeric features that resultin similar charge/shape features as compared to the native helices.

[0006] Peptides, or compounds with similar charge/shape features andhaving the activity of the peptides described herein, can be identifiedby phage display using wild-type amphipathic helix and a mutantamphipathic helix peptides as positive and negative selectors,respectively.

[0007] The compositions or agents of the invention may comprise, consistessentially of, or consist of the peptide sequences disclosed herein.The phrase “consists essentially of or consisting essentially of” or thelike, when applied to anti-HCV peptides encompassed by the presentinvention refers to peptide sequences like those disclosed herein, butwhich contain additional amino acids (or analogs or derivatives thereofas discussed above). Such additional amino acids, etc., however, do notmaterially affect the basic and novel characteristic(s) of thesepeptides in modulating, attenuating, or inhibiting HCV infection,replication, and/or pathogenesis, including the specific quantitativeeffects of these peptides, compared to those of the correspondingpeptides disclosed herein.

[0008] In one approach, the agent may be a transdominant inhibitor ofthe membrane association function whereby forms of the amphipathic helixthat interfere with the ability of the helix to form oligomers can beused. Thus, by generating or providing a mutant form of the helixcontaining one or more amino acid substutions, this form may associatewith the native helix to provide an inactive form or rendering it unableto dimerize or oligomerize with additional native forms. In oneapproach, the decoy peptide is mutated to convert hydrophobic aminoacids to hydrophilic ones thus destroying the hydrophobic face of thehelix. For example, mutated versions of the peptide sequence for NS5Astrains would include SGSWLRD D WDW E CEVLSD D KTWLKAK (SEQ ID NO: 15)or SGSWLRD D WDW E CTVLTD D KTWLQSKL. (SEQ ID NO: 16)

[0009] SEQ ID NOS: 15 and 16 are used as PEP2 in Example 4, below.

[0010] In another approach, the agent is a competitive inhibitor of theamphipathic helix. These competitive inhibitors may interrupt thebinding between the helix and the membrane, achieving the desiredeffect. Such inhibitors may be fragments of the wild type sequence ofthe amphipathic sequence or variants or mutants thereof. Fragments ofthe HCV nonstructural proteins that may be used as competitiveinhibitors may include, but are not limited to:YIEQGMMLAEQFKQKALGLLQTASRHAEV, (NS4B 6-34) (SEQ ID NO: 462)QDVLKEVKAAASKVKANLLSVEE, (NS5B 65-87) (SEQ ID NO: 3) andDVRCHARKAVAHINSVWKD. (NS5B 107-125) (SEQ ID NO: 4)

[0011] Another competitive inhibitor of NS5A would include:SGSWLRDVWDWICTVLTDFKTWLQSKL (SEQ ID NO: 14) and variants and mutantsthereof. Variants and mutants of this peptide would include a peptidewith one or more of the following amino acid substitutions: substitutionof L at amino acid 16 by A or K; or substitution of the T at amino acid17 by A; or substitution of the D at amino acid 18 by A; or substitutionof the F at amino acid 19 by A; or substitution of the K at amino acid20 by A; or substitution of the W at amino acid 22 by A; or substitutionof the L at amino acid 23 by K, and derivatives thereof. Another suchmutant of NS5A is: SGSX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀QSK L, where X₁ is W, A or F; X₂ is L, A or K; X₃ is R, A orN; X₄ is D, A or S;X₅ is V, I, D or S; X₆ is W, A or F; X₇ is D, A or S;X₈ is W,A or F; X₉ is I, E or L; X₁₀ is C, A or S; X₁₁ is T, E, A or W;X₁₂ is V, D or S; X₁₃ is L, A or K; X₁₄is T, S, A or W; X₁₅is D, A or S;X₁₆is F,D, or L; X₁₇is K, A or L; X₁₈ is T, A or W; X₁₉ is W, A or F;and X₂₀ is L, A or K. In another embodiment, X₁, X₂, X₄, X₆, X₈, X₁₀,X₁₃, X₁₅, X₁₆ or X₂₀may be D or X₁₆ is A. Where the agent is acompetitive inhibitor, some of the above mutations would enhance theinhibitory activity of the peptide, others would completely or partiallyabolish the inhibitory activity of the peptide. Mutations may beobserved alone or in combination, for example, any one substitution,X₁₋₂₀, occurs within the context of the wild type sequence at any onetime, a combination of two mutations such as: X₅ and X₉, X₅ and X₁₆, orX₉ and X₁₆ or three mutations are found in the same peptide, X₅, withX₉, and X₁₆. Additional mutations of the above inhibitor may include thesequence: X₁X₂DVWDWICTX₃X₄X₅X₆X₇X₈X₉, wherein when X₁ and X₂ arepresent, X₁ is L and X₂ is R; wherein X₁ and X₂ are present, X₃, X₄, X₅,X₆, X₇, X₈ and X₉ are optionally present, and when X₃, X₄, X₅, X₆, X₇,X₈ and X₉ are present, X₃ is V, X₄ is L, X₅ is T, X₆ is D, X₇ is F, X₈is K and X₉ is T; and wherein X₆ is present, X₃, X₄ and X₅ are allpresent, wherein X₆ is D, X₃ is V, X₄ is L, and X₅ is T. In particularwhere the sequence is LRDVWDWICTVLTDFKT (SEQ ID NO: 463), LRDVWDWICT(SEQ ID NO: 464) or DVWDWICTVLTD (SEQ ID NO: 465). In anotherembodiment, the competitive inhibitor may be SWLRDVWDWIC (SEQ ID NO:466).

[0012] A mutant, and competitive inhibitor, of NS4B may include thesequence: X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉ where X₁ is Y, A or H; X₂ is I, E or L;X₃ is E, A or Q; X₄ is Q, V or R; X₅ is G, A or D; X₆ is M, E or C; X₇is M, E or C; X₈ is L, E or Q; X₉ is A, D or S; X₁₀ is E, A or Q; X₁₁ isQ, V, or R; X₁₂ is F, D or L; X₁₃ is K, I or Q; X₁₄ is Q, V, or R; X₁₅is K, I or Q; X₁₆ is A, D or G; X₁₇ is L, A or K; X₁₈ is G, E or S; X₁₉is L, A or K; X₂₀ is L, A or K; X₂₁ is Q, V or R; X₂₂ is T, A or W; X₂₃is A, D or G; X₂₄ is S, D or A; X₂₅ is R, L or W; X₂₆ is Q, V or R; X₂₇is A, D or G; and X₂₈ is E, A or Q.

[0013] While the majority of the above mutations and substitutions areconservative (i.e. wild type hydrophobic residues are substituted withadditional hydrophobic residues (for example, A to F), charged residuesare substituted with similarly charged residues, etc.), it is noted thatnonconservative substitutions may also be performed. For example, wherea wild type hydrophobic residue is substituted with a hydrophilicresidue, or a negatively charged residue (for example, D) is substitutedwith a positively charged residue (for example, K).

[0014] Where the agent is a competitive inhibitor, some of the abovemutations would enhance the inhibitory activity of the peptide, otherswould completely or partially abolish the inhibitory activity of thepeptide. Mutations may be observed alone or in combination, for example,any one substitution, X₁₋₂₈, occurs within the context of the wild typesequence at any one time, a combination of two mutations such as: X₂ andX₅, X₂ and X₁₉, or X₅ and X₁₉ or three mutations are found in the samepeptide, X₂, with X₅, and X₁₉.

[0015] In another embodiment, the agent may be a complementary peptideto the helix. Complementary peptides may interrupt the binding betweenthe helix and the membrane, achieving the desired effect. Suchcomplementary peptides may also inhibit the formation of the amphipathichelix, may interact or bind to the helix to inhibit binding of the helixto cellular membranes, or may otherwise inhibit the amphipathic helices.

[0016] For example, the HCV genomic RNA sequence that codes for the NS4Bamphipathic helix in the HCV genotype 1A sequence is:UACAUCGAGCAAGGGAUGAUGCUCGCTGAGCAGU (SEQ ID NO: 17)UCAAGCAGAAGGCCCUCGGCCUCCUGCAGACCGC GUCCCGCCAAGCAGAG.

[0017] (Kolykhalov, A. A. and Rice, C. M., Science 277 (5325), 570-574(1997); GenBank Accession No.: AF009606.) The protein translation of theabove sequence is: YIEQGMMLAEQFKQKALGLLQTASRQAE (SEQ ID NO: 18). Thereverse complement cDNA sequence corresponding to the HCV genomic RNAsequence that codes for the NS4B amphipathic helix (GenBank AccessionNo.: AF009606) is: CTCTGCTTGGCGGGACGCGGTCTGCAGGAGGCCGAGGGCCTTCTGCTTGAACTGCTCAGCGAGCATCATCCCTTGCTCGATGTA (SEQ ID NO: 19). The complementarypeptide translated from the reverse complement sequence is:LCLAGRGLQEAEGLLLELLSEHHPLLDV (SEQ ID NO: 20). This complementarypeptide, as a whole, or in part, may be active as an HCV antiviral ormay be useful in the prediction of small molecules that are HCVantivirals. In one embodiment a fragment of this complementary peptidecomprising 6-27 amino acids may be used in the discovery of HCVantivirals. Table 1 sets forth exemplary peptides (SEQ ID NOS: 21-295)of this embodiment. TABLE 1 All smaller NS4B complementary peptides with6 or more amino acids: Sequence Sequence Identification Number LQEAEGSEQ ID NO: 21 CLAGRG SEQ ID NO: 22 LELLSE SEQ ID NO: 23 GLQEAE SEQ IDNO: 24 LLELLS SEQ ID NO: 25 QEAEGL SEQ ID NO: 26 RGLQEA SEQ ID NO: 27LAGRGL SEQ ID NO: 28 GRGLQE SEQ ID NO: 29 ELLSEH SEQ ID NO: 30 LLLELLSEQ ID NO: 31 LCLAGR SEQ ID NO: 32 HPLLDV SEQ ID NO: 33 AEGLLL SEQ IDNO: 34 AGRGLQ SEQ ID NO: 35 EAEGLL SEQ ID NO: 36 LSEHHP SEQ ID NO: 37HHPLLD SEQ ID NO: 38 EGLLLE SEQ ID NO: 39 SEHHPL SEQ ID NO: 40 LLSEHHSEQ ID NO: 41 EHHPLL SEQ ID NO: 42 GLLLEL SEQ ID NO: 43 LELLSEH SEQ IDNO: 44 ELLSEHH SEQ ID NO: 45 LLELLSE SEQ ID NO: 46 LLSEHHP SEQ ID NO: 47HHPLLDV SEQ ID NO: 48 GLLLELL SEQ ID NO: 49 LAGRGLQ SEQ ID NO: 50LSEHHPL SEQ ID NO: 51 LCLAGRG SEQ ID NO: 52 LQEAEGL SEQ ID NO: 53AGRGLQE SEQ ID NO: 54 AEGLLLE SEQ ID NO: 55 GRGLQEA SEQ ID NO: 56RGLQEAE SEQ ID NO: 57 QEAEGLL SEQ ID NO: 58 SEHHPLL SEQ ID NO: 59EHHPLLD SEQ ID NO: 60 GLQEAEG SEQ ID NO: 61 EGLLLEL SEQ ID NO: 62LLLELLS SEQ ID NO: 63 EAEGLLL SEQ ID NO: 64 CLAGRGL SEQ ID NO: 65CLAGRGLQ SEQ ID NO: 66 QEAEGLLL SEQ ID NO: 67 LAGRGLQE SEQ ID NO: 68LCLAGRGL SEQ ID NO: 69 AGRGLQEA SEQ ID NO: 70 LLLELLSE SEQ ID NO: 71LELLSEHH SEQ ID NO: 72 AEGLLLEL SEQ ID NO: 73 EGLLLELL SEQ ID NO: 74LLSEHHPL SEQ ID NO: 75 GRGLQEAE SEQ ID NO: 76 EAEGLLLE SEQ ID NO: 77RGLQEAEG SEQ ID NO: 78 LLELLSEH SEQ ID NO: 79 SEHHPLLD SEQ ID NO: 80LSEHHPLL SEQ ID NO: 81 LQEAEGLL SEQ ID NO: 82 ELLSEHHP SEQ ID NO: 83GLLLELLS SEQ ID NO: 84 EHHPLLDV SEQ ID NO: 85 GLQEAEGL SEQ ID NO: 86LLSEHHPLL SEQ ID NO: 87 LELLSEHHP SEQ ID NO: 88 RGLQEAEGL SEQ ID NO: 89ELLSEHHPL SEQ ID NO: 90 LCLAGRGLQ SEQ ID NO: 91 AGRGLQEAE SEQ ID NO: 92GLLLELLSE SEQ ID NO: 93 LSEHHPLLD SEQ ID NO: 94 EGLLLELLS SEQ ID NO: 95GLQEAEGLL SEQ ID NO: 96 AEGLLLELL SEQ ID NO: 97 QEAEGLLLE SEQ ID NO: 98SEHHPLLDV SEQ ID NO: 99 LLLELLSEH SEQ ID NO: 100 GRGLQEAEG SEQ ID NO:101 CLAGRGLQE SEQ ID NO: 102 EAEGLLLEL SEQ ID NO: 103 LAGRGLQEA SEQ IDNO: 104 LLELLSEHH SEQ ID NO: 105 LQEAEGLLL SEQ ID NO: 106 LSEHHPLLDV SEQID NO: 107 LELLSEHHPL SEQ ID NO: 108 RGLQEAEGLL SEQ ID NO: 109LLSEHHPLLD SEQ ID NO: 110 AEGLLLELLS SEQ ID NO: 111 GLQEAEGLLL SEQ IDNO: 112 EGLLLELLSE SEQ ID NO: 113 LCLAGRGLQE SEQ ID NO: 114 LLLELLSEHHSEQ ID NO: 115 EAEGLLLELL SEQ ID NO: 116 GLLLELLSEH SEQ ID NO: 117ELLSEHHPLL SEQ ID NO: 118 GRGLQEAEGL SEQ ID NO: 119 LQEAEGLLLE SEQ IDNO: 120 LLELLSEHHP SEQ ID NO: 121 CLAGRGLQEA SEQ ID NO: 122 QEAEGLLLELSEQ ID NO: 123 AGRGLQEAEG SEQ ID NO: 124 LAGRGLQEAE SEQ ID NO: 125LLELLSEHHPL SEQ ID NO: 126 CLAGRGLQEAE SEQ ID NO: 127 RGLQEAEGLLL SEQ IDNO: 128 LAGRGLQEAEG SEQ ID NO: 129 LQEAEGLLLEL SEQ ID NO: 130LLLELLSEHHP SEQ ID NO: 131 ELLSEHHPLLD SEQ ID NO: 132 AGRGLQEAEGL SEQ IDNO: 133 LLSEHHPLLDV SEQ ID NO: 134 LCLAGRGLQEA SEQ ID NO: 135GLLLELLSEHH SEQ ID NO: 136 LELLSEHHPLL SEQ ID NO: 137 QEAEGLLLELL SEQ IDNO: 138 EGLLLELLSEH SEQ ID NO: 139 GRGLQEAEGLL SEQ ID NO: 140AEGLLLELLSE SEQ ID NO: 141 GLQEAEGLLLE SEQ ID NO: 142 EAEGLLLELLS SEQ IDNO: 143 EGLLLELLSEHH SEQ ID NO: 144 RGLQEAEGLLLE SEQ ID NO: 145GLLLELLSEHHP SEQ ID NO: 146 LQEAEGLLLELL SEQ ID NO: 147 GLQEAEGLLLEL SEQID NO: 148 LAGRGLQEAEGL SEQ ID NO: 149 GRGLQEAEGLLL SEQ ID NO: 150QEAEGLLLELLS SEQ ID NO: 151 AEGLLLELLSEH SEQ ID NO: 152 AGRGLQEAEGLL SEQID NO: 153 LELLSEHHPLLD SEQ ID NO: 154 LCLAGRGLQEAE SEQ ID NO: 155LLLELLSEHHPL SEQ ID NO: 156 LLELLSEHHPLL SEQ ID NO: 157 ELLSEHHPLLDV SEQID NO: 158 EAEGLLLELLSE SEQ ID NO: 159 CLAGRGLQEAEG SEQ ID NO: 160CLAGRGLQEAEGL SEQ ID NO: 161 RGLQEAEGLLLEL SEQ ID NO: 162 LLELLSEHHPLLDSEQ ID NO: 163 GRGLQEAEGLLLE SEQ ID NO: 164 LCLAGRGLQEAEG SEQ ID NO: 165LELLSEHHPLLDV SEQ ID NO: 166 EGLLLELLSEHHP SEQ ID NO: 167 EAEGLLLELLSEHSEQ ID NO: 168 GLQEAEGLLLELL SEQ ID NO: 169 AGRGLQEAEGLLL SEQ ID NO: 170AEGLLLELLSEHH SEQ ID NO: 171 LAGRGLQEAEGLL SEQ ID NO: 172 QEAEGLLLELLSESEQ ID NO: 173 LLLELLSEHHPLL SEQ ID NO: 174 LQEAEGLLLELLS SEQ ID NO: 175GLLLELLSEHHPL SEQ ID NO: 176 EAEGLLLELLSEHH SEQ ID NO: 177EGLLLELLSEHHPL SEQ ID NO: 178 AGRGLQEAEGLLLE SEQ ID NO: 179LCLAGRGLQEAEGL SEQ ID NO: 180 GLLLELLSEHHPLL SEQ ID NO: 181QEAEGLLLELLSEH SEQ ID NO: 182 RGLQEAEGLLLELL SEQ ID NO: 183LLLELLSEHHPLLD SEQ ID NO: 184 LQEAEGLLLELLSE SEQ ID NO: 185LLELLSEHHPLLDV SEQ ID NO: 186 GLQEAEGLLLELLS SEQ ID NO: 187GRGLQEAEGLLLEL SEQ ID NO: 188 AEGLLLELLSEHHP SEQ ID NO: 189CLAGRGLQEAEGLL SEQ ID NO: 190 LAGRGLQEAEGLLL SEQ ID NO: 191LLLELLSEHHPLLDV SEQ ID NO: 192 AGRGLQEAEGLLLEL SEQ ID NO: 193AEGLLLELLSEHHPL SEQ ID NO: 194 GLLLELLSEHHPLLD SEQ ID NO: 195LQEAEGLLLELLSEH SEQ ID NO: 196 GLQEAEGLLLELLSE SEQ ID NO: 197LCLAGRGLQEAEGLL SEQ ID NO: 198 EAEGLLLELLSEHHP SEQ ID NO: 199GRGLQEAEGLLLELL SEQ ID NO: 200 QEAEGLLLELLSEHH SEQ ID NO: 201LAGRGLQEAEGLLLE SEQ ID NO: 202 CLAGRGLQEAEGLLL SEQ ID NO: 203EGLLLELLSEHHPLL SEQ ID NO: 204 RGLQEAEGLLLELLS SEQ ID NO: 205LAGRGLQEAEGLLLEL SEQ ID NO: 206 GLLLELLSEHHLPLLDV SEQ ID NO: 207GRGLQEAEGLLLELLS SEQ ID NO: 208 AGRGLQEAEGLLLELL SEQ ID NO: 209GLQEAEGLLLELLSEH SEQ ID NO: 210 RGLQEAEGLLLELLSE SEQ ID NO: 211LQEAEGLLLELLSEHH SEQ ID NO: 212 CLAGRGLQEAEGLLLE SEQ ID NO: 213QEAEGLLLELLSEHHP SEQ ID NO: 214 AEGLLLELLSEHHPLL SEQ ID NO: 215EAEGLLLELLSEHHPL SEQ ID NO: 216 LCLAGRGLQEAEGLLL SEQ ID NO: 217EGLLLELLSEHHPLLD SEQ ID NO: 218 LCLAGRGLQEAEGLLLE SEQ ID NO: 219QEAEGLLLELLSEHHPL SEQ ID NO: 220 EAEGLLLELLSEHHPLL SEQ ID NO: 221RGLQEAEGLLLELLSEH SEQ ID NO: 222 CLAGRGLQEAEGLLLEL SEQ ID NO: 223GRGLQEAEGLLLELLSE SEQ ID NO: 224 LAGRGLQEAEGLLLELL SEQ ID NO: 225AGRGLQEAEGLLLELLS SEQ ID NO: 226 GLQEAEGLLLELLSEHH SEQ ID NO: 227EGLLLELLSEHHPLLDV SEQ ID NO: 228 AEGLLLELLSEHHPLLD SEQ ID NO: 229LQEAEGLLLELLSEHHP SEQ ID NO: 230 CLAGRGLQEAEGLLLELL SEQ ID NO: 231LQEAEGLLLELLSEHHPL SEQ ID NO: 232 GRGLQEAEGLLLELLSEH SEQ ID NO: 233EAEGLLLELLSEHHPLLD SEQ ID NO: 234 AEGLLLELLSEHHPLLDV SEQ ID NO: 235GLQEAEGLLLELLSEHHP SEQ ID NO: 236 RGLQEAEGLLLELLSEHH SEQ ID NO: 237QEAEGLLLELLSEHHPLL SEQ ID NO: 238 LAGRGLQEAEGLLLELLS SEQ ID NO: 239LCLAGRGLQEAEGLLLEL SEQ ID NO: 240 AGRGLQEAEGLLLELLSE SEQ ID NO: 241AGRGLQEAEGLLLELLSEH SEQ ID NO: 242 GLQEAEGLLLELLSEHHPL SEQ ID NO: 243GRGLQEAEGLLLELLSEHH SEQ ID NO: 244 LCLAGRGLQEAEGLLLELL SEQ ID NO: 245CLAGRGLQEAEGLLLELLS SEQ ID NO: 246 RGLQEAEGLLLELLSEHHP SEQ ID NO: 247LAGRGLQEAEGLLLELLSE SEQ ID NO: 248 QEAEGLLLELLSEHHPLLD SEQ ID NO: 249LQEAEGLLLELLSEHHPLL SEQ ID NO: 250 EAEGLLLELLSEHHPLLDV SEQ ID NO: 251RGLQEAEGLLLELLSEHHPL SEQ ID NO: 252 GLQEAEGLLLELLSEHHPLL SEQ ID NO: 253AGRGLQEAEGLLLELLSEHH SEQ ID NO: 254 LAGRGLQEAEGLLLELLSEH SEQ ID NO: 255GRGLQEAEGLLLELLSEHHP SEQ ID NO: 256 LCLAGRGLQEAEGLLLELLS SEQ ID NO: 257LQEAEGLLLELLSEHHPLLD SEQ ID NO: 258 QEAEGLLLELLSEHHPLLDV SEQ ID NO: 259CLAGRGLQEAEGLLLELLSE SEQ ID NO: 260 LCLAGRGLQEAEGLLLELLSE SEQ ID NO: 261RGLQEAEGLLLELLSEHHPLL SEQ ID NO: 262 LQEAEGLLLELLSEHHPLLDV SEQ ID NO:263 GRGLQEAEGLLLELLSEHHPL SEQ ID NO: 264 AGRGLQEAEGLLLELLSEHHP SEQ IDNO: 265 LAGRGLQEAEGLLLELLSEHH SEQ ID NO: 266 CLAGRGLQEAEGLLLELLSEH SEQID NO: 267 GLQEAEGLLLELLSEHHPLLD SEQ ID NO: 268 GLQEAEGLLLELLSEHHPLLDVSEQ ID NO: 269 AGRGLQEAEGLLLELLSEHHPL SEQ ID NO: 270CLAGRGLQEAEGLLLELLSEHH SEQ ID NO: 271 GRGLQEAEGLLLELLSEHHPLL SEQ ID NO:272 RGLQEAEGLLLELLSEHHPLLD SEQ ID NO: 273 LAGRGLQEAEGLLLELLSEHHP SEQ IDNO: 274 LCLAGRGLQEAEGLLLELLSEH SEQ ID NO: 275 CLAGRGLQEAEGLLLELLSEHHPSEQ ID NO: 276 GRGLQEAEGLLLELLSEHHPLLD SEQ ID NO: 277LCLAGRGLQEAEGLLLELLSEHH SEQ ID NO: 278 LAGRGLQEAEGLLLELLSEHHPL SEQ IDNO: 279 RGLQEAEGLLLELLSEHHPLLDV SEQ ID NO: 280 AGRGLQEAEGLLLELLSEHHPLLSEQ ID NO: 281 AGRGLQEAEGLLLELLSEHHPLLD SEQ ID NO: 282CLAGRGLQEAEGLLLELLSEHHPL SEQ ID NO: 283 LCLAGRGLQEAEGLLLELLSEHHP SEQ IDNO: 284 GRGLQEAEGLLLELLSEHHPLLDV SEQ ID NO: 285 LAGRGLQEAEGLLLELLSEHHPLLSEQ ID NO: 286 AGRGLQEAEGLLLELLSEHHPLLDV SEQ ID NO: 287LCLAGRGLQEAEGLLLELLSEHHPL SEQ ID NO: 288 LAGRGLQEAEGLLLELLSEHHPLLD SEQID NO: 289 CLAGRGLQEAEGLLLELLSEHHPLL SEQ ID NO: 290LCLAGRGLQEAEGLLLELLSEHHPLL SEQ ID NO: 291 LAGRGLQEAEGLLLELLSEHHPLLDV SEQID NO: 292 CLAGRGLQEAEGLLLELLSEHHPLLD SEQ ID NO: 293LCLAGRGLQEAEGLLLELLSEHHPLLD SEQ ID NO: 294 CLAGRGLQEAEGLLLELLSEHHPLLDVSEQ ID NO: 295

[0018] In a particular embodiment, the complementary peptides of theinvention are used, in whole or in part, in the prediction of smallmolecules that are HCV antivirals. “Small molecule” as defined above,may include synthetic organic structures typical of pharmaceuticals, andmay also include peptides, nucleic acids, peptide nucleic acids,carbohydrates, lipids, and the like. Additionally, small molecules, asused herein, may include chemically synthesized peptidomimetics of the6-mer to 9-mer peptides of the invention.

[0019] Additionally, the HCV genomic RNA sequence that codes for theNS5A amphipathic helix in the HCV genotype 1A sequence is:UGGCUAAGGGACAUCUGGGACUGGAUAUGCGAGGUGCUGAGCGACUUUAAGACCUGGCUGAAAGCCAAGCUC (SEQ ID NO: 296). (Kolykhalov, A. A. and Rice,C. M., Science 277 (5325), 570-574 (1997); GenBank Accession No.:AF009606.) The protein translation of the above sequence is:WLRDIWDWICEVLSDFKTWLKAKL (SEQ ID NO: 297). The reverse complement cDNAsequence corresponding to the HCV genomic RNA sequence that codes forthe NS5A amphipathic helix (GenBank Accession No.: AF009606) is:GAGCTTGGCTTTCAGCCAGGTCTTAAAGTCGCTCAGCACCTCGCATATCCAGTCCCAGATGTCCCTTAGCCA (SEQ ID NO: 298). This reverse complement sequencecontains a stop codon at codon 23. The complementary peptide translatedfrom the reverse complement sequence is: ELGFQPGLKVAQHLAYPVPDVP (SEQ IDNO: 299). This complementary peptide, as a whole, or in part, may beactive as an HCV antiviral or may be useful in the prediction of smallmolecules that are HCV antivirals. In one embodiment a fragment of thiscomplementary peptide comprising 6-21 amino acids may is used in thediscovery of HCV antivirals. Table 2 sets forth exemplary peptides (SEQID NOS: 300-451) of this embodiment. TABLE 2 NS5A complementary peptideswith 6 or more amino acids: Sequence Sequence Identification NumberAQHLAY SEQ ID NO: 300 LGFQPG SEQ ID NO: 301 GLKVAQ SEQ ID NO: 302 ELGFQPSEQ ID NO: 303 PVPDVP SEQ ID NO: 304 FQPGLK SEQ ID NO: 305 GFQPGL SEQ IDNO: 306 VAQHLA SEQ ID NO: 307 LKVAQH SEQ ID NO: 308 HLAYPV SEQ ID NO:309 KVAQHL SEQ ID NO: 310 QPGLKV SEQ ID NO: 311 LAYPVP SEQ ID NO: 312YPVPDV SEQ ID NO: 313 QHLAYP SEQ ID NO: 314 PGLKVA SEQ ID NO: 315 AYPVPDSEQ ID NO: 316 QPGLKVA SEQ ID NO: 317 ELGFQPG SEQ ID NO: 318 HLAYPVP SEQID NO: 319 LAYPVPD SEQ ID NO: 320 AQHLAYP SEQ ID NO: 321 FQPGLKV SEQ IDNO: 322 KVAQHLA SEQ ID NO: 323 LGFQPGL SEQ ID NO: 324 AYPVPDV SEQ ID NO:325 PGLKVAQ SEQ ID NO: 326 LKVAQHL SEQ ID NO: 327 GLKVAQH SEQ ID NO: 328VAQHLAY SEQ ID NO: 329 GFQPGLK SEQ ID NO: 330 QHLAYPV SEQ ID NO: 331YPVPDVP SEQ ID NO: 332 AYPVPDVP SEQ ID NO: 333 PGLKVAQH SEQ ID NO: 334QHLAYPVP SEQ ID NO: 335 LAYPVPDV SEQ ID NO: 336 LKVAQHLA SEQ ID NO: 337GLKVAQHL SEQ ID NO: 338 GFQPGLKV SEQ ID NO: 339 AQHLAYPV SEQ ID NO: 340VAQHLAYP SEQ ID NO: 341 KVAQHLAY SEQ ID NO: 342 ELGFQPGL SEQ ID NO: 343LGFQPGLK SEQ ID NO: 344 FQPGLKVA SEQ ID NO: 345 HLAYPVPD SEQ ID NO: 346QPGLKVAK SEQ ID NO: 347 ELGFQPGLK SEQ ID NO: 348 VAQHLAYPV SEQ ID NO:349 LGFQPGLKV SEQ ID NO: 350 QPGLKVAQH SEQ ID NO: 351 KVAQHLAYP SEQ IDNO: 352 PGLKVAQHL SEQ ID NO: 353 FQPGLKVAQ SEQ ID NO: 354 GLKVAQHLA SEQID NO: 355 LAYPVPDVP SEQ ID NO: 356 HLAYPVPDV SEQ ID NO: 357 GFQPGLKVASEQ ID NO: 358 AQHLAYPVP SEQ ID NO: 359 QHLAYPVPD SEQ ID NO: 360LKVAQHLAY SEQ ID NO: 361 QPGLKVAQHL SEQ ID NO: 362 PGLKVAQHLA SEQ ID NO:363 HLAYPVPDVP SEQ ID NO: 364 GLKVAQHLAY SEQ ID NO: 365 VAQHLAYPVP SEQID NO: 366 KVAQHLAYPV SEQ ID NO: 367 GFQPGLKVAQ SEQ ID NO: 368QHLAYPVPDV SEQ ID NO: 369 AQHLAYPVPD SEQ ID NO: 370 FQPGLKVAQH SEQ IDNO: 371 ELGFQPGLKV SEQ ID NO: 372 LKVAQHLAYP SEQ ID NO: 373 LGFQPGLKVASEQ ID NO: 374 QHLAYPVPDVP SEQ ID NO: 375 LKVAQHLAYPV SEQ ID NO: 376VAQHLAYPVPD SEQ ID NO: 377 PGLKVAQHLAY SEQ ID NO: 378 LGFQPGLKVAQ SEQ IDNO: 379 KVAQHLAYPVP SEQ ID NO: 380 ELGFQPGLKVA SEQ ID NO: 381GLKVAQHLAYP SEQ ID NO: 382 AQHLAYPVPDV SEQ ID NO: 383 FQPGLKVAQHL SEQ IDNO: 384 QPGLKVAQHLA SEQ ID NO: 385 GFQPGLKVAQH SEQ ID NO: 386LGFQPGLKVAQH SEQ ID NO: 387 PGLKVAQHLAYP SEQ ID NO: 388 LKVAQHLAYPVP SEQID NO: 389 GFQPGLKVAQHL SEQ ID NO: 390 VAQHLAYPVPDV SEQ ID NO: 391QPGLKVAQHLAY SEQ ID NO: 392 ELGFQPGLKVAQ SEQ ID NO: 393 AQHLAYPVPDVP SEQID NO: 394 KVAQHLAYPVPD SEQ ID NO: 395 FQPGLKVAQHLA SEQ ID NO: 396GLKVAQHLAYPV SEQ ID NO: 397 LGFQPGLKVAQHL SEQ ID NO: 398 VAQHLAYPVPDVPSEQ ID NO: 399 PGLKVAQHLAYPV SEQ ID NO: 400 GFQPGLKVAQHLA SEQ ID NO: 401LKVAQHLAYPVPD SEQ ID NO: 402 QPGLKVAQHLAYP SEQ ID NO: 403 ELGFQPGLKVAQHSEQ ID NO: 404 GLKVAQHLAYPVP SEQ ID NO: 405 FQPGLKVAQHLAY SEQ ID NO: 406KVAQHLAYPVPDV SEQ ID NO: 407 ELGFQPGLKVAQHL SEQ ID NO: 408QPGLKVAQHLAYPV SEQ ID NO: 409 GFQPGLKVAQHLAY SEQ ID NO: 410LGFQPGLKVAQHLA SEQ ID NO: 411 FQPGLKVAQHLAYP SEQ ID NO: 412KVAQHLAYPVPDVP SEQ ID NO: 413 PGLKVAQHLAYPVP SEQ ID NO: 414LKVAQHLAYPVPDV SEQ ID NO: 415 GLKVAQHLAYPVPD SEQ ID NO: 416GLKVAQHLAYPVPDV SEQ ID NO: 417 PGLKVAQHLAYPVPD SEQ ID NO: 418ELGFQPGLKVAQHLA SEQ ID NO: 419 LGFQPGLKVAQHLAY SEQ ID NO: 420GFQPGLKVAQHLAYP SEQ ID NO: 421 QPGLKVAQHLAYPVP SEQ ID NO: 422FQPGLKVAQHLAYPV SEQ ID NO: 423 LKVAQHLAYPVPDVP SEQ ID NO: 424FQPGLKVAQHLAYPVP SEQ ID NO: 425 GFQPGLKVAQHLAYPV SEQ ID NO: 426GLKVAQHLAYPVPDVP SEQ ID NO: 427 PGLKVAQHLAYPVPDV SEQ ID NO: 428QPGLKVAQHLAYPVPD SEQ ID NO: 429 ELGFQPGLKVAQHLAY SEQ ID NO: 430LGFQPGLKVAQHLAYP SEQ ID NO: 431 GFQPGLKVAQHLAYPVP SEQ ID NO: 432FQPGLKVAQHLAYPVPD SEQ ID NO: 433 QPGLKVAQHLAYPVPDV SEQ ID NO: 434PGLKVAQHLAYPVPDVP SEQ ID NO: 435 LGFQPGLKVAQHLAYPV SEQ ID NO: 436ELGFQPGLKVAQHLAYP SEQ ID NO: 437 ELGFQPGLKVAQHLAYPV SEQ ID NO: 438QPGLKVAQHLAYPVPDVP SEQ ID NO: 439 LGFQPGLKVAQHLAYPVP SEQ ID NO: 440FQPGLKVAQHLAYPVPDV SEQ ID NO: 441 GFQPGLKVAQHLAYPVPD SEQ ID NO: 442FQPGLKVAQHLAYPVPDVP SEQ ID NO: 443 LGFQPGLKVAQHLAYPVPD SEQ ID NO: 444GFQPGLKVAQHLAYPVPDV SEQ ID NO: 445 ELGFQPGLKVAQHLAYPVP SEQ ID NO: 446LGFQPGLKVAQHLAYPVPDV SEQ ID NO: 447 ELGFQPGLKVAQHLAYPVPD SEQ ID NO: 448GFQPGLKVAQHLAYPVPDVP SEQ ID NO: 449 ELGFQPGLKVAQHLAYPVPDV SEQ ID NO: 450LGFQPGLKVAQHLAYPVPDVP SEQ ID NO: 451

[0020] The peptides that mimic the helices and functional fragmentsthereof are administered in formulations and by routes well understood.A variety of methods for introducing such substances are known,typically, by injection, aerosol administration, suppository, and, withproper design, oral administration. This general statement is true aswell with respect to providing expression systems for peptidesrepresented by the helix mimics.

[0021] In addition to the peptides or other compounds of the invention,combination therapies including known HCV inhibitors can be utilized inthe present invention. For example, it may be desirable to administerboth a peptide or peptides of the invention in combination withinterferon to a subject infected with HCV. Other drugs or compoundsknown in the art to be effective against HCV, can also be used.

[0022] In one embodiment, the invention provides a method of screeningcompounds, to identify those that selectively inhibit the binding of HCVnonstructural proteins (for example, NS4B or NS5A) and cellularmembranes. Methods known to those of skill in the art, can be readilyadapted to detect interference with the binding of these components. Themethod of screening may involve high-throughput techniques. For example,to screen for compounds that selectively inhibit the binding of HCVnonstructural proteins and cellular membranes, a synthetic reaction mix,a viral fragment or component, or a preparation of any thereof,comprising an HCV nonstructural protein and a labeled substrate orligand of such polypeptide is incubated in the absence or the presenceof a candidate molecule that may inhibit the binding of the HCVnonstructural proteins and the cellular membrane. The ability of thecandidate molecule to inhibit the binding of the HCV nonstructuralprotein and the cellular membrane is reflected in decreased binding ofthe labeled ligand or decreased production of product from suchsubstrate.

[0023] In another aspect, the screening can be performed by adding thecandidate compound to intact cells that have been infected by HCV, orthat contain an HCV replicon, and then examining the component ofinterest to demonstrate the effect on this component, or the effect onviral or replicon replication. An exemplary cell-based in vitro assayfor this purpose is disclosed in PCT International Publication WO02/18369. Alternatively, the screening can be performed by adding thetest agent to in vitro translation reactions and then proceeding withthe established analysis. As another alternative, purified or partiallypurified components which have been determined to interact with oneanother by the methods described above can be placed under conditions inwhich the interaction between them would normally occur, with andwithout the addition of the candidate compound, and the procedurespreviously established to analyze the interaction can be used to assessthe impact of the candidate compound. However their anti-HCV activity isinitially assayed, peptide or other inhibitors of the present inventionshould cause inhibition of infection, replication, or pathogenesis ofHepatitis C Virus in vitro or in vivo when introduced into a host cellcontaining the virus, and exhibit an IC₅₀ in the range of from about0.0001 nM to 100 μM in an in vitro assay for at least one step ininfection, replication, or pathogenesis of HCV, more preferably fromabout 0.0001 nM to 75 μM, more preferably from about 0.0001 nM to 50 μM,more preferably from about 0.0001 nM to 25 μM, more preferably fromabout 0.0001 nM to 10 μM, and even more preferably from about 0.0001 nMto 1 μM.

[0024] In another embodiment of the invention, the method of screeningmay be by phage display. A method of obtaining selective ligands thatbind a chosen target is to select from a library of proteins or peptidesdisplayed on phage. In order to obtain a novel binding protein against achosen target, such as an amphipathic helix region of an HCV component,DNA molecules, each encoding a protein or peptide fragment thereof, anda structural signal calling for the display of the protein on the outersurface of a chosen genetic package (bacterial cell, bacterial spore orphage) are introduced into a genetic package. The protein is expressedand the potential binding domain is displayed on the outer surface ofthe package. The package is then exposed to the target. If the geneticpackage binds to the target, then it is confirmed that the correspondingbinding domain is indeed displayed by the genetic package. Packageswhich display the binding domain (and thereby bind the target) areseparated from those which do not. For example, in the presentinvention, the target may be the amphipathic helix or a mutatedamphipathic helix. Potential peptides, which may then be used asanti-HCV agents, are screened by determination of which will bind to theamphipathic helix or a mutated amphipathic helix. Preferred peptides arethose that not only bind to the amphipathic helix, but in addition,block or inhibit the amphipathic helix from binding to its receptor orbinding site, thereby inhibiting infectivity. Examples of peptidesidentified by phage display are set forth in Table 3. TABLE 3 Peptidesidentified by phage display as ligands of the amphipathic helix of NS5A:Sequence Sequence Identification Number HDSFANATGRFWP SEQ ID NO: 454QGTSPSRLAVPLA SEQ ID NO: 455 ISSKTGMSSEPPS SEQ ID NO: 456 ILSSIDALGSDSHSEQ ID NO: 457 LDDRSVPTVISQR SEQ ID NO: 458 YPSKPGNVTPKAP SEQ ID NO: 459QAQGERALK SEQ ID NO: 460 TDKRASPLTVQAR SEQ ID NO: 461

[0025] In all of the embodiments of the invention, the active agentwhich will interact, generally, either with the sites on the cytoplasmicmembrane to which the amphipathic helix binds or will interact with theamphipathic helix itself, may be derivatized or coupled to additionalcomponents. By “derivatives” of these agents is meant modificationsthereof that do not interfere with their ability to interact with thesites or the helix, but may optionally confer some additional usefulproperty. One particularly useful property is the ability to penetratecell membranes, and preferred among the derivatives or conjugated formsare those coupled to such facilitators. An additional desired coupledcomponent may be a labeling component such as a fluorescent dye, anenzyme, or a radioisotope. One particularly useful label is, forexample, green fluorescent protein in any of its many colors and forms.Green fluorescent protein thus, includes, not only forms of thisfluorescent protein that fluoresce green, but also those that fluorescevarious other colors such as red and blue. These forms of the proteinare commercially available as are recombinant materials for theirproduction.

[0026] The compositions of the invention can be administered asconventional HCV therapeutics. The compositions of the invention mayinclude more than one ingredient which interrupts the binding of theamphipathic helix to the membranes and more than one peptide of theinvention.

[0027] The precise formulations and modes of administration of theanti-HCV compositions of the invention will depend on the nature of theanti-HCV agent, the condition of the subject, and the judgment of thepractitioner. Design of such administration and formulation is routineoptimization generally carried out without difficulty by thepractitioner.

[0028] In addition to the assay methods, methods to identify compoundsor protocols against HCV infection, and methods to treat HCV infectionsas set forth above, the invention provides compositions which areeffective to elicit immunological responses to HCV in appropriatesubjects, such as humans or other animals subject to HCV infection.

[0029] Administration may be performed using conventional methods,typically by injection. The elicited immunological response is helpfulin general HCV prophylaxis.

[0030] The following examples are intended to illustrate but not tolimit the invention.

EXAMPLES Example 1 Effect of Helix Disruption on Membrane Association

[0031] The bottom panels of FIGS. 3A-3C show the structures of therelevant portion of NS5A used in this example. FIG. 3A shows the aminoacid sequence and helix formed at positions 4-27 in the NS5A protein ofHepatitis C virus genotype I a. FIG. 3B shows the deletion of positions7-27, thus deleting the helix, as shown by the brackets. This deletionwas conducted by PCR mutagenesis essentially as described by Glenn, J.S., et al., J. Virol (1998) 72:9303-9306 on plasmid pBRTM/HCV 827-3011which encodes the HCV nonstructural proteins as described by Grakoui,A., et al., J. Virol. (1993) 67:1385-1395. This vector encodes the NSproteins under the T7 promoter and encephalomyocarditis internalribosome entry site control elements and directs the synthesis andprocessing of proteins NS3, NS4A, NS4B, NS5A and NS5B. FIG. 3C shows amutant which was obtained using PCR mutagenesis as described above usinga primer (5′-TCCGGCTCCTGGCTAAGGGAC GA CTGGGA- (SEQ ID NO: 452) CTGG GAATGCGAGGTGCTGAGCGAC GA TAAGAC- C-3′)

[0032] in which codons isoleucine-8, isoleucine-12, and phenylalanine-19of NS5A were changed to encode aspartate, glutamate, and aspartate,respectively. This results in disruption of the hydrophobic region ofthe helix.

[0033] Each of these plasmids was expressed and distribution of the NSSAprotein, which is produced by the plasmids in cells, was observed. Aliver derived cell line, Huh-7, was first infected with recombinantvaccinia virus encoding T7-RNA polymerase and then transfected withpBRTM/HCV 827-3001, or by this plasmid modified as described in FIGS. 3Band 3C.

[0034] After incubation to effect protein production, the cells werefixed with formaldehyde and immunostained with a monoclonal antibodyagainst NS5A and a Texas Red-labeled donkey anti-mouse secondaryantibody essentially as described by Glenn, J. S., et al., J. Virol.(1990) 64:3104-3107. As shown in the upper panel of FIG. 3A, perinuclearpunctate vesicular staining suggestive of a Golgi-like intracellulardistribution pattern was readily observed, as was, occasionally, asomewhat reticulin chicken-wire-like staining pattern, characteristic ofthe ER. Both patterns have been reported previously when NS5A isexpressed either alone or in combination with other HCV NS proteinsusing a variety of expression systems. See, for example, Kim, J. E., etal., Arch. Virol. (1999) 144:329-343; Huang, Y., et al., Biochem.Biophys. Res. Commun. (2001) 281:732-740.

[0035] However, when the construct of FIG. 3B, containing a deletedportion of NS5A was expressed, as shown in the upper panel of FIG. 3B,cytoplasmic membrane localization was abolished and a nuclearlocalization pattern was obtained. A cryptic nuclear localization signalin the NS5A C-terminal domain has previously been reported by Ide, Y.,et a., Gene (1996) 182:203-211.

[0036] When the construct described in FIG. 3C, which disrupts thehydrophobic face of the helix, is expressed in these cells, again, asshown in the upper panel of FIG. 3C, the cytoplasmic membranelocalization pattern was lost and localization to the nucleus wasobserved.

[0037]FIGS. 3A-3C show these results at two magnification levels. Theuppermost panel in each case shows results for a single cell and theintermediate panel shows results for a number of cells. As shown in theintermediate panels in FIGS. 3B and 3C, occasionally distribution of thedye throughout the cell was observed rather than localization to thenucleus. However, even in these cases, no specific localization to thecytoplasmic membrane could be detected.

Example 2 Intracellular Location of GFP-Labeled Helix

[0038] Three constructs were made. First, as a control, the HCV encodingsequences of pBRTM/HCV 827-3011 were replaced with the coding sequencefor jellyfish Aequorea Victoria green fluorescent protein (GFP) using aPCR cloned insert from plasmid C109 which contains E-GFP (Clontech), toobtain “T7-GFP.” A fragment of the NS5A gene encoding the amphipathichelix was fused into frame with the sequence encoding the N-terminus ofGFP in T7-GFP by creating a common PstI site using PCR mutagenesis. Thispermits junction of the first 31 amino acids of NS5A to the 5′ side ofserine-2 of the GFP-encoding sequence in T7-GFP. The resulting vectorwas labeled “T7-5AGFP.” A similar vector, designated “T7-5ANHGFP” wasconstructed in a manner similar to “T7-5AGFP” except that thecorresponding segment from the mutated form of NS5A set forth in FIG. 3Cwas employed in place of the wildtype helix. Thus, “T7-5ANHGFP” issimilar to “T7-5AGFP” except that in the NS5A helix, Ile at position 8is converted to Asp, Ile at position 12 is converted to Glu, and Phe atposition 19 is converted to Asp.

[0039] These constructs were used to effect expression of GFP or of theGFP fusions in Huh-7 cells as described in Example I and thedistribution of fluorescence was evaluated with the results as shown inFIGS. 4A-4C. As shown in FIG. 4A, cells expressing GFP alone show a widediffuse pattern including concentration in the nucleus. Cells expressing5AGFP show distribution of fluorescence similar to that of the NS5Aprotein itself with a Golgi-like intracellular distribution pattern andER staining. Cells expressing 5ANHGFP failed to show this pattern butinstead mimic the distribution pattern of GFP.

Example 3 Effect of Helix Disruption on HCV RNA Replication

[0040] Comparative high efficiency, second generation, bicistronic,subgenomic RNA replicons of HCV described by Blight, K. J., et al.,Science (2001) 290:1972-1974 were constructed with a neomycin-resistancegene. Similar constructs were made with wildtype NS5A and with NS5Aaltered in the amphipathic helix as described in Examples 1 and 2.Diagrams of these constructs are shown in the upper panels of FIGS. 5Aand 5B.

[0041] The construct containing the wildtype NS5A, Bart79I, was made byPCR mutagenesis of HCVreplbBartMan/AvaII (Blight, supra) such thatnucleotide 5336 was changed from a G to T resulting in a change in NS5Acodon 1179 from serine to isoleucine. This mutation results in adramatic increase in replication efficiency of the HCV subgenomicreplicon. Bart5X79I (containing the modified helix) was made by PCRmutagenesis of Bart79I using a primer (5′-G A TTGGGATTGG GAATGCACGGTGTTGACT (SEQ ID NO: 453) GAT GA CAAGACCTGG-3′)

[0042] in which codons valine-8, isoleucine-12, and phenylalanine-19 ofNS5A were changed to encode aspartate, glutamate, and aspartate,respectively. All mutations were confirmed by DNA sequencing.

[0043] The RNA replication efficiency was then assayed by ability toestablish G418-resistant colonies after transfection of Huh-7 cells asfollows: Replicon RNA's were prepared by in vitro transcription withT7-RNA polymerase of ScaI-linearized plasmids (Bart79I or Bart5X79I)followed by DNase treatment and purification. Replicons were thenelectroporated into Huh-7 cells and neomycin-resistant colonies selectedby inclusion of 1 mg/ml G418 in the culture media. Methods wereessentially as described by Blight (supra). Colonies were detected bystaining with cresyl violet. Multiple independent preparations ofreplicons and replication assays yielded similar results.

[0044] As shown in FIG. 5A, lower panel, the replicon with wildtype NS5AN-terminus gave rise to numerous colonies while disrupting theamphipathic nature of the N-terminal helix results in a dramaticinhibition of HCV genome replication as shown in the lower panel of FIG.5B.

[0045] There was no decreased transfection efficiency or any decrease inthe ability to establish colonies when control experiments usingplasmids encoding drug resistance marker along with only the wildtype ormutant NS5A proteins were used in place of the replicons above, thusestablishing that the results were not due to an increased cytotoxicityassociated with mutant NS5A.

Example 4 Floatation Assay

[0046] In this membrane floatation assay, the ability of the NS5Aamphipathic helix to bind to a preparation of microsomal membranes wastested. NS5A proteins, both the NS5A wildtype or mutant amphipathichelix (described in Example 1), were in vitro translated and[³⁵S]-labeled using the Promega TNT reticulocyte lysate kit. PlasmidspC5A or pC5ANH were used as transcription templates. Aliquots of thereactions were combined with a membrane fraction derived from Huh-7cells, with or without synthetic peptides, and overlayed with a 5-40%OptiPrep step gradient. After centrifugation for 4 hrs at 40,000×g in aSW60 rotor, 500 ml fractions were collected from the top and theproteins in each gradient fraction were precipitated and analyzed bySDS-PAGE. The percentage of protein “floating” to the top of thegradient with membranes (fraction 2 from the top) was then quantifiedusing a Molecular Dynamics phosphorimager.

[0047]FIGS. 6A and 6B show a comparison of the results for wildtype andmutant forms. The numbers correspond to Optiprep gradient (5-40%)fraction number. Non-membrane associated proteins remain at the bottom(right side) of the gradient, whereas membranes—and associatedproteins—“float” towards less dense gradient fractions present at thetop (left side). As shown, in the reaction mixture containing wildtype,the synthesized NS5A floated toward the top of the gradient coupled withthe membrane. This was not found in the case of the mutant form. In bothcases, there was a degree of non-specific binding associated withfractions that settle to the bottom of the gradient. An advantage ofthis method is elimination of the artifacts caused by non-specificbinding, as illustrated by the labeling of high density fractions inboth mixtures.

[0048]FIG. 6C shows the percentage of NS5A wildtype or mutant proteinthat float with the membrane fractions as a percentage of the totalsynthesized. As seen, 25% of the wildtype, but only 0.8% of the mutant,was associated with the membrane.

[0049]FIGS. 7A and 7B show the results of a similar floatationexperiment in the presence and absence of a test peptide that is acompetitive inhibitor of the NS5A helix. This peptide contains aminoacids 1-27 of NS5A with a C-terminal “flag” DYKD. The assay described inthe preceding paragraph was repeated in the presence and absence of thispeptide. As shown in FIG. 7A, the percentage of NS5A floated in theabsence of peptide is arbitrarily normalized to 100%; in comparison withthis, the presence of the test peptide lowered the percentage to about21%.

[0050] Similarly, the assay was modified by using, in place of NS5A, afusion protein consisting of the amphipathic helix of NS5A with greenfluorescent protein. As can be seen in FIG. 7B, the amount of labeledNS5A floated with the membrane was set in the absence of test peptide at100%. In comparison, the presence of the test peptide lowered thepercentage floated to about 21%.

[0051] Moreover, as shown in FIG. 6C, a synthetic peptide designed tomimic the wild type AH not only competitively inhibited membraneassociation of NSSA, but did so in a dose-dependent manner. The maximalextent of inhibition achieved pharmacologically was comparable to thatobtained by genetic mutation of the AH. In addition, the syntheticpeptide appears equally effective against NS5A derived from differentgenotypes (data not shown), including those most refractory to currenttherapies. This convenient membrane floatation assay has also provenwell-suited for current efforts focused on studying the mechanisticdetails of the AH membrane-targeting domain, and ideal for guidingongoing development of peptidomimetic compounds designed to resemble keyelements, or bind to specific features, of the AH. Such compoundsrepresent an exciting potential addition to current anti-HCV combinationtherapy regimens.

[0052]FIG. 8 shows Pharmacologic inhibition of NS5A membraneassociation. Huh-7 cells-derived membranes were treated with PEPI (apeptide mimicking the wild type amphipathic helix of NS5A), PEP2 (acontrol peptide) or mock-treated with water. PEP1 (a peptide thatcorresponds to the wild type N-terminal amphipathic helix of NS5A (aminoacids 1-26) with a C-terminal FLAG tag) and PEP2 (which is identical toPEP1 except amino acids isoleucine-8, isoleucine-12, andphenylalanine-19 of NS5A were changed to aspartate, glutamate, andaspartate, respectively) were synthesized by AnaSpec Inc. In-vitrotranslated NS5A (or NS5ANH) was incubated with membranes and variousconcentrations of peptides or water prior to analysis by membranefloatation assays set forth above. The amount of NS5A floating with nopeptide treatment typically is˜30% of the total in-vitro translatedNS5A. For comparison between treatment conditions, results werenormalized to the no-treatment control. The percent of NS5A (or NS5ANH)floating with the membranes under the indicated conditions wasdetermined as in FIG. 6A and expressed relative to mock-treated control.Error bars represent standard error of the mean.

[0053] While the invention has been described in detail with referenceto certain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

[0054] References

[0055] 1. Araga S, Blalock J E. Use of complementary peptides and theirantibodies in B-cell-mediated autoimmune disease: prevention ofexperimental autoimmune myasthenia gravis with a peptide vaccine.Immunomethods. 1994 October;5(2): 130-5.

[0056] 2. Araga S, Galin F S, Kishimoto M, Adachi A, Blalock J B.Prevention of experimental autoimmune myasthenia gravis by a monoclonalantibody to a complementary peptide for the main immunogenic region ofthe acetylcholine receptors. J Immunol. 1996 Jul. 1;157(1):386-92.

[0057] 3. Araga S, LeBoeuf R D, Blalock J E. Prevention of experimentalautoimmune myasthenia gravis by manipulation of the immune network witha complementary peptide for the acetylcholine receptor. Proc Natl AcadSci U S A. 1993 Sep. 15;90(18):8747-51.

[0058] 4. Blalock J E, Bost K L. Binding of peptides that are specifiedby complementary RNAs. Biochem J. 1986 Mar. 15;234(3):679-83.

[0059] 5. Blalock J E, Whitaker J N, Benveniste E N, Bost K L. Use ofpeptides encoded by complementary RNA for generating anti-idiotypicantibodies of predefined specificity. Methods Enzymol. 1989;178:63-74.

[0060] 6. Bost K L, Blalock J E. Complementary peptides as interactivesites for protein binding. Viral Immunol. 1989 Winter;2(4):229-38.

[0061] 7. Bost K L, Blalock J E. Preparation and use of complementarypeptides. Methods Enzymol. 1989;168:16-28.

[0062] 8. Bost K L, Blalock J E. Production of anti-idiotypic antibodiesby immunization with a pair of complementary peptides. J Mol Recognit.1989 April;1 (4):179-83.

[0063] 9. Bost K L, Smith E M, Blalock J E. Similarity between thecorticotropin (ACTH) receptor and a peptide encoded by an RNA that iscomplementary to ACTH mRNA. Proc Natl Acad Sci U S A. 1985March;82(5):1372-5.

[0064] 10. Hawiger, J., Noninvasive intracellular delivery of functionalpeptides and proteins. Current Opinion in Chemical Biology, 1999. 3: p.89-94.

[0065] 11. Holsworth D D, Kiely J S, Root-Bernstein R S, Overhiser R W.Antisense-designed peptides: a comparative study focusing on possiblecomplements to angiotensin II. Pept Res. 1994 July-August;7(4):185-93.

[0066] 12. Kolykhalov, A. A. and Rice,C. M. (Science 277 (5325), 570-574(1997).

[0067] 13. Maria Lindgren, M. H., Alain Prochiantz, and Ulo Langel,Cell-penetrating peptides. Trends in Pharmacological Sciences, 2000. 21:p. 99-103.

[0068] 14. May C. Morris, J. D., Jean Mery, Frederic Heitz and GillesDivita, A peptide carrier for the delivery of biologically activeproteins into mammalian cells. Nature Biotechnology, 2001. 19: p.1173-1176.

[0069] 15. Pfister R R, Haddox J L, Blalock J E, Sommers C I, Coplan L,Villain M. Synthetic complementary peptides inhibit a neutrophilchemoattractant found in the alkali-injured cornea. Comea. 2000May;19(3):384-9.

[0070] 16. Mauricio Rojas, J. P. D., Zhongjia Tan, and Yao-Zhong Lin,Genetic engineering of proteins with cell membrane permeability. NatureBiotechnology, 1998.16: p. 370-375.

[0071] 17. Anne Scheller, B. W., Matthias Meizig, Michael Bienert, andJohannes Oehlke, Evidence for an amphipathicity independent cellularuptake of amphipathic cell-penetrating peptides. European Journal ofBiochemistry, 2000. 267: p. 6043-6049.

[0072] 18. Swords B H, Carr D J, Blalock J E, Berecek K H. An antibodydirected against a peptide encoded by RNA complementary to mRNA forvasopressin recognizes putative vasopressin receptors.Neuroendocrinology. 1990 April;51 (4):487-92.

[0073] 19. Weigent D A, Clarke B L, Blalock J E. Peptide design using agenetically patterned binary code: growth hormone-releasing hormone as amodel. Immunomethods. 1994 October;5(2):91-7.

[0074] 20. Xue Yan Liu, D. R., Ruth Ann Veach, Danya Liu, SheilaTimmons, Robert D. Collins, and Jacek Hawiger, Peptide-directedsuppression of a pro-inflammatory cytokine response. The Journal ofBiological Chemistry, 2000. 275: p. 16774-16778.

[0075] 21. Yunfeng Zhao, D. L., John Burkett, Heinz Kohler, Chemicalengineering of cell penetrating antibodies. Journal of ImmunologicalMethods, 2001. 254: p. 137-145.

[0076] 22. Lianshan Zhang, T. R. T., Xue-Yan Liu, Sheila Timmons, Ann D.Colosia, Jacek Hawiger and James P. Tam, Preparation of functionallyactive cell-permeable peptides by single-step ligation of two peptidemodules. Proc. Natl. Acad. Sci. USA, 1998. 95: p. 9184-9189.

[0077] 23. Badkar, A., Talluri, K., Tenjarla, S., Jaynes, J., Banga, A.,In Vitro Release Testing of a Peptide Gel. Pharm. Technol., 44-52(January 2000).

[0078] 24. Bernkop-Schnurch, A. Chitosan and its derivatives: potentialexcipients for peroral peptide delivery systems. Int. J. Pharm. 194,1-13 (2000).

[0079] 25. Gonda, I., The Ascent of Pulmonary Drug Delivery. J. Pahrm.Sci. 89, 940-945 (2000).

[0080] 26. Jung, T. Kamm, W., Breitenbach, A., Kaiserling, E., Xiao, J.X., Kissel, T., Biodegradable nonoparticles for oral delivery ofpeptides: is there a role for polymers to affect mucosal uptake? Eur. J.Pharm. Biopharm. 50, 147-160 (2000).

[0081] 27. Latham, P., Therapeutic Peptides Revisited. Nat. Biotech. 17,755-757 (1999).

[0082] 28. Pillai, O., Nair, V., Poduri, R., Pancchagnula, R.,Transdermal Iontophoresis. Part II: Peptide and Protein Delivery.Methods Find. Exp. Clin. Pharmacol. 21(3): 229-240 (1999).

[0083] 29. Sakuma, S. Hayashi, M., Akashi, M., Design of nanoparticlescomposed of graft copolymers for oral peptide delivery. Advanced DrugDelivery Reviews 47, 21-37 (2001).

[0084] 30. Takeuchi, H., Yamamoto, H., Kawashima, Y. Mucoadhesivenanoparticulate systeme for peptide drug delivery. Advanced DrugDelivery Reviews 47, 39-51 (2001).

[0085] 31. Veronese, F. M., Morpugo, M., Bioconjugation inpharmaceuticalchemistry. II Farnaco 54, 497-516 (1999).

[0086] 32. Veuillex, F., Kalia, Y. N., Jacques, Y., Dashusses, J., Buri,P., Factors and strategies for improving buccal absorption of peptides.Eur. J. Pharm. Biopharm. 51, 93-109 (2001).

[0087] 33. Wang, W., Jiang, J., Ballard, C. E., Wang, B., ProdrugApproaches to the Improved Delivery of Peptide Drugs. Cur. Pharm. Des.5, 265-287 (1999).

1 470 1 28 PRT Hepatitis C virus 1 Ile Glu Gln Gly Met Met Leu Ala GluGln Phe Lys Gln Lys Ala Leu 1 5 10 15 Gly Leu Leu Gln Thr Ala Ser ArgHis Ala Glu Val 20 25 2 21 PRT Hepatitis C virus 2 Leu Arg Asp Ile TrpAsp Trp Ile Cys Glu Val Leu Ser Asp Phe Lys 1 5 10 15 Thr Trp Leu LysAla 20 3 23 PRT Hepatitis C virus 3 Gln Asp Val Leu Lys Glu Val Lys AlaAla Ala Ser Lys Val Lys Ala 1 5 10 15 Asn Leu Leu Ser Val Glu Glu 20 419 PRT Hepatitis C virus 4 Asp Val Arg Cys His Ala Arg Lys Ala Val AlaHis Ile Asn Ser Val 1 5 10 15 Trp Lys Asp 5 24 PRT Hepatitis C virus 5Trp Leu Arg Asp Val Trp Asp Trp Ile Cys Thr Val Leu Thr Asp Phe 1 5 1015 Lys Thr Trp Leu Gln Ser Lys Leu 20 6 24 PRT Hepatitis C virus 6 TrpLeu Arg Asp Val Trp Asp Trp Val Cys Thr Ile Leu Thr Asp Phe 1 5 10 15Lys Asn Trp Leu Thr Ser Lys Leu 20 7 24 PRT Hepatitis C virus 7 Trp LeuArg Asp Ile Trp Glu Trp Val Leu Ser Ile Leu Thr Asp Phe 1 5 10 15 LysAsn Trp Leu Ser Ala Lys Leu 20 8 24 PRT Hepatitis C virus 8 Trp Leu ArgIle Ile Trp Asp Trp Val Cys Ser Val Val Ser Asp Phe 1 5 10 15 Lys ThrTrp Leu Ser Ala Lys Ile 20 9 24 PRT Hepatitis C virus 9 Trp Leu Arg ThrIle Trp Asp Trp Val Cys Ser Val Leu Ala Asp Phe 1 5 10 15 Lys Ala TrpLeu Ser Ala Lys Ile 20 10 24 PRT Hepatitis C virus 10 Trp Leu His AspIle Trp Asp Trp Val Cys Ile Val Leu Ser Asp Phe 1 5 10 15 Lys Thr TrpLeu Ser Ala Lys Ile 20 11 24 PRT Hepatitis C virus 11 Trp Leu Trp AspVal Trp Asp Trp Val Leu His Val Leu Ser Asp Phe 1 5 10 15 Lys Thr CysLeu Lys Ala Lys Phe 20 12 24 PRT Hepatitis C virus 12 Trp Leu Tyr AspIle Val Asn Trp Val Cys Thr Val Leu Ala Asp Phe 1 5 10 15 Lys Leu TrpLeu Gly Ala Lys Ile 20 13 24 PRT Hepatitis C virus 13 Trp Leu Arg AspIle Trp Asp Trp Val Cys Thr Val Leu Ser Asp Phe 1 5 10 15 Arg Val TrpLeu Lys Ser Lys Leu 20 14 27 PRT Hepatitis C virus 14 Ser Gly Ser TrpLeu Arg Asp Val Trp Asp Trp Ile Cys Thr Val Leu 1 5 10 15 Thr Asp PheLys Thr Trp Leu Gln Ser Lys Leu 20 25 15 26 PRT Artificial sequenceMutated version of non-structural protein NS5A 15 Ser Gly Ser Trp LeuArg Asp Asp Trp Asp Trp Glu Cys Glu Val Leu 1 5 10 15 Ser Asp Asp LysThr Trp Leu Lys Ala Lys 20 25 16 27 PRT Artificial sequence Mutatedversion of non-structural protein NS5A 16 Ser Gly Ser Trp Leu Arg AspAsp Trp Asp Trp Glu Cys Thr Val Leu 1 5 10 15 Thr Asp Asp Lys Thr TrpLeu Gln Ser Lys Leu 20 25 17 84 RNA Hepatitis C virus 17 uacaucgagcaagggaugau gcucgcugag caguucaagc agaaggcccu cggccuccug 60 cagaccgcgucccgccaagc agag 84 18 28 PRT Hepatitis C virus 18 Tyr Ile Glu Gln GlyMet Met Leu Ala Glu Gln Phe Lys Gln Lys Ala 1 5 10 15 Leu Gly Leu LeuGln Thr Ala Ser Arg Gln Ala Glu 20 25 19 84 DNA Hepatitis C virus 19ctctgcttgg cgggacgcgg tctgcaggag gccgagggcc ttctgcttga actgctcagc 60gagcatcatc ccttgctcga tgta 84 20 28 PRT Hepatitis C virus 20 Leu Cys LeuAla Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu 1 5 10 15 Glu LeuLeu Ser Glu His His Pro Leu Leu Asp Val 20 25 21 6 PRT Hepatitis C virus21 Leu Gln Glu Ala Glu Gly 1 5 22 6 PRT Hepatitis C virus 22 Cys Leu AlaGly Arg Gly 1 5 23 6 PRT Hepatitis C virus 23 Leu Glu Leu Leu Ser Glu 15 24 6 PRT Hepatitis C virus 24 Gly Leu Gln Glu Ala Glu 1 5 25 6 PRTHepatitis C virus 25 Leu Leu Glu Leu Leu Ser 1 5 26 6 PRT Hepatitis Cvirus 26 Gln Glu Ala Glu Gly Leu 1 5 27 6 PRT Hepatitis C virus 27 ArgGly Leu Gln Glu Ala 1 5 28 6 PRT Hepatitis C virus 28 Leu Ala Gly ArgGly Leu 1 5 29 6 PRT Hepatitis C virus 29 Gly Arg Gly Leu Gln Glu 1 5 306 PRT Hepatitis C virus 30 Glu Leu Leu Ser Glu His 1 5 31 6 PRTHepatitis C virus 31 Leu Leu Leu Glu Leu Leu 1 5 32 6 PRT Hepatitis Cvirus 32 Leu Cys Leu Ala Gly Arg 1 5 33 6 PRT Hepatitis C virus 33 HisPro Leu Leu Asp Val 1 5 34 6 PRT Hepatitis C virus 34 Ala Glu Gly LeuLeu Leu 1 5 35 6 PRT Hepatitis C virus 35 Ala Gly Arg Gly Leu Gln 1 5 366 PRT Hepatitis C virus 36 Glu Ala Glu Gly Leu Leu 1 5 37 6 PRTHepatitis C virus 37 Leu Ser Glu His His Pro 1 5 38 6 PRT Hepatitis Cvirus 38 His His Pro Leu Leu Asp 1 5 39 6 PRT Hepatitis C virus 39 GluGly Leu Leu Leu Glu 1 5 40 6 PRT Hepatitis C virus 40 Ser Glu His HisPro Leu 1 5 41 6 PRT Hepatitis C virus 41 Leu Leu Ser Glu His His 1 5 426 PRT Hepatitis C virus 42 Glu His His Pro Leu Leu 1 5 43 6 PRTHepatitis C virus 43 Gly Leu Leu Leu Glu Leu 1 5 44 7 PRT Hepatitis Cvirus 44 Leu Glu Leu Leu Ser Glu His 1 5 45 7 PRT Hepatitis C virus 45Glu Leu Leu Ser Glu His His 1 5 46 7 PRT Hepatitis C virus 46 Leu LeuGlu Leu Leu Ser Glu 1 5 47 7 PRT Hepatitis C virus 47 Leu Leu Ser GluHis His Pro 1 5 48 7 PRT Hepatitis C virus 48 His His Pro Leu Leu AspVal 1 5 49 7 PRT Hepatitis C virus 49 Gly Leu Leu Leu Glu Leu Leu 1 5 507 PRT Hepatitis C virus 50 Leu Ala Gly Arg Gly Leu Gln 1 5 51 7 PRTHepatitis C virus 51 Leu Ser Glu His His Pro Leu 1 5 52 7 PRT HepatitisC virus 52 Leu Cys Leu Ala Gly Arg Gly 1 5 53 7 PRT Hepatitis C virus 53Leu Gln Glu Ala Glu Gly Leu 1 5 54 7 PRT Hepatitis C virus 54 Ala GlyArg Gly Leu Gln Glu 1 5 55 7 PRT Hepatitis C virus 55 Ala Glu Gly LeuLeu Leu Glu 1 5 56 7 PRT Hepatitis C virus 56 Gly Arg Gly Leu Gln GluAla 1 5 57 7 PRT Hepatitis C virus 57 Arg Gly Leu Gln Glu Ala Glu 1 5 587 PRT Hepatitis C virus 58 Gln Glu Ala Glu Gly Leu Leu 1 5 59 7 PRTHepatitis C virus 59 Ser Glu His His Pro Leu Leu 1 5 60 7 PRT HepatitisC virus 60 Glu His His Pro Leu Leu Asp 1 5 61 7 PRT Hepatitis C virus 61Gly Leu Gln Glu Ala Glu Gly 1 5 62 7 PRT Hepatitis C virus 62 Glu GlyLeu Leu Leu Glu Leu 1 5 63 7 PRT Hepatitis C virus 63 Leu Leu Leu GluLeu Leu Ser 1 5 64 7 PRT Hepatitis C virus 64 Glu Ala Glu Gly Leu LeuLeu 1 5 65 7 PRT Hepatitis C virus 65 Cys Leu Ala Gly Arg Gly Leu 1 5 668 PRT Hepatitis C virus 66 Cys Leu Ala Gly Arg Gly Leu Gln 1 5 67 8 PRTHepatitis C virus 67 Gln Glu Ala Glu Gly Leu Leu Leu 1 5 68 8 PRTHepatitis C virus 68 Leu Ala Gly Arg Gly Leu Gln Glu 1 5 69 8 PRTHepatitis C virus 69 Leu Cys Leu Ala Gly Arg Gly Leu 1 5 70 8 PRTHepatitis C virus 70 Ala Gly Arg Gly Leu Gln Glu Ala 1 5 71 8 PRTHepatitis C virus 71 Leu Leu Leu Glu Leu Leu Ser Glu 1 5 72 8 PRTHepatitis C virus 72 Leu Glu Leu Leu Ser Glu His His 1 5 73 8 PRTHepatitis C virus 73 Ala Glu Gly Leu Leu Leu Glu Leu 1 5 74 8 PRTHepatitis C virus 74 Glu Gly Leu Leu Leu Glu Leu Leu 1 5 75 8 PRTHepatitis C virus 75 Leu Leu Ser Glu His His Pro Leu 1 5 76 8 PRTHepatitis C virus 76 Gly Arg Gly Leu Gln Glu Ala Glu 1 5 77 8 PRTHepatitis C virus 77 Glu Ala Glu Gly Leu Leu Leu Glu 1 5 78 8 PRTHepatitis C virus 78 Arg Gly Leu Gln Glu Ala Glu Gly 1 5 79 8 PRTHepatitis C virus 79 Leu Leu Glu Leu Leu Ser Glu His 1 5 80 8 PRTHepatitis C virus 80 Ser Glu His His Pro Leu Leu Asp 1 5 81 8 PRTHepatitis C virus 81 Leu Ser Glu His His Pro Leu Leu 1 5 82 8 PRTHepatitis C virus 82 Leu Gln Glu Ala Glu Gly Leu Leu 1 5 83 8 PRTHepatitis C virus 83 Glu Leu Leu Ser Glu His His Pro 1 5 84 8 PRTHepatitis C virus 84 Gly Leu Leu Leu Glu Leu Leu Ser 1 5 85 8 PRTHepatitis C virus 85 Glu His His Pro Leu Leu Asp Val 1 5 86 8 PRTHepatitis C virus 86 Gly Leu Gln Glu Ala Glu Gly Leu 1 5 87 9 PRTHepatitis C virus 87 Leu Leu Ser Glu His His Pro Leu Leu 1 5 88 9 PRTHepatitis C virus 88 Leu Glu Leu Leu Ser Glu His His Pro 1 5 89 9 PRTHepatitis C virus 89 Arg Gly Leu Gln Glu Ala Glu Gly Leu 1 5 90 9 PRTHepatitis C virus 90 Glu Leu Leu Ser Glu His His Pro Leu 1 5 91 9 PRTHepatitis C virus 91 Leu Cys Leu Ala Gly Arg Gly Leu Gln 1 5 92 9 PRTHepatitis C virus 92 Ala Gly Arg Gly Leu Gln Glu Ala Glu 1 5 93 9 PRTHepatitis C virus 93 Gly Leu Leu Leu Glu Leu Leu Ser Glu 1 5 94 9 PRTHepatitis C virus 94 Leu Ser Glu His His Pro Leu Leu Asp 1 5 95 9 PRTHepatitis C virus 95 Glu Gly Leu Leu Leu Glu Leu Leu Ser 1 5 96 9 PRTHepatitis C virus 96 Gly Leu Gln Glu Ala Glu Gly Leu Leu 1 5 97 9 PRTHepatitis C virus 97 Ala Glu Gly Leu Leu Leu Glu Leu Leu 1 5 98 9 PRTHepatitis C virus 98 Gln Glu Ala Glu Gly Leu Leu Leu Glu 1 5 99 9 PRTHepatitis C virus 99 Ser Glu His His Pro Leu Leu Asp Val 1 5 100 9 PRTHepatitis C virus 100 Leu Leu Leu Glu Leu Leu Ser Glu His 1 5 101 9 PRTHepatitis C virus 101 Gly Arg Gly Leu Gln Glu Ala Glu Gly 1 5 102 9 PRTHepatitis C virus 102 Cys Leu Ala Gly Arg Gly Leu Gln Glu 1 5 103 9 PRTHepatitis C virus 103 Glu Ala Glu Gly Leu Leu Leu Glu Leu 1 5 104 9 PRTHepatitis C virus 104 Leu Ala Gly Arg Gly Leu Gln Glu Ala 1 5 105 9 PRTHepatitis C virus 105 Leu Leu Glu Leu Leu Ser Glu His His 1 5 106 9 PRTHepatitis C virus 106 Leu Gln Glu Ala Glu Gly Leu Leu Leu 1 5 107 10 PRTHepatitis C virus 107 Leu Ser Glu His His Pro Leu Leu Asp Val 1 5 10 10810 PRT Hepatitis C virus 108 Leu Glu Leu Leu Ser Glu His His Pro Leu 1 510 109 10 PRT Hepatitis C virus 109 Arg Gly Leu Gln Glu Ala Glu Gly LeuLeu 1 5 10 110 10 PRT Hepatitis C virus 110 Leu Leu Ser Glu His His ProLeu Leu Asp 1 5 10 111 10 PRT Hepatitis C virus 111 Ala Glu Gly Leu LeuLeu Glu Leu Leu Ser 1 5 10 112 10 PRT Hepatitis C virus 112 Gly Leu GlnGlu Ala Glu Gly Leu Leu Leu 1 5 10 113 10 PRT Hepatitis C virus 113 GluGly Leu Leu Leu Glu Leu Leu Ser Glu 1 5 10 114 10 PRT Hepatitis C virus114 Leu Cys Leu Ala Gly Arg Gly Leu Gln Glu 1 5 10 115 10 PRT HepatitisC virus 115 Leu Leu Leu Glu Leu Leu Ser Glu His His 1 5 10 116 10 PRTHepatitis C virus 116 Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu 1 5 10 11710 PRT Hepatitis C virus 117 Gly Leu Leu Leu Glu Leu Leu Ser Glu His 1 510 118 10 PRT Hepatitis C virus 118 Glu Leu Leu Ser Glu His His Pro LeuLeu 1 5 10 119 10 PRT Hepatitis C virus 119 Gly Arg Gly Leu Gln Glu AlaGlu Gly Leu 1 5 10 120 10 PRT Hepatitis C virus 120 Leu Gln Glu Ala GluGly Leu Leu Leu Glu 1 5 10 121 10 PRT Hepatitis C virus 121 Leu Leu GluLeu Leu Ser Glu His His Pro 1 5 10 122 10 PRT Hepatitis C virus 122 CysLeu Ala Gly Arg Gly Leu Gln Glu Ala 1 5 10 123 10 PRT Hepatitis C virus123 Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu 1 5 10 124 10 PRT HepatitisC virus 124 Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly 1 5 10 125 10 PRTHepatitis C virus 125 Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu 1 5 10 12611 PRT Hepatitis C virus 126 Leu Leu Glu Leu Leu Ser Glu His His Pro Leu1 5 10 127 11 PRT Hepatitis C virus 127 Cys Leu Ala Gly Arg Gly Leu GlnGlu Ala Glu 1 5 10 128 11 PRT Hepatitis C virus 128 Arg Gly Leu Gln GluAla Glu Gly Leu Leu Leu 1 5 10 129 11 PRT Hepatitis C virus 129 Leu AlaGly Arg Gly Leu Gln Glu Ala Glu Gly 1 5 10 130 11 PRT Hepatitis C virus130 Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu 1 5 10 131 11 PRTHepatitis C virus 131 Leu Leu Leu Glu Leu Leu Ser Glu His His Pro 1 5 10132 11 PRT Hepatitis C virus 132 Glu Leu Leu Ser Glu His His Pro Leu LeuAsp 1 5 10 133 11 PRT Hepatitis C virus 133 Ala Gly Arg Gly Leu Gln GluAla Glu Gly Leu 1 5 10 134 11 PRT Hepatitis C virus 134 Leu Leu Ser GluHis His Pro Leu Leu Asp Val 1 5 10 135 11 PRT Hepatitis C virus 135 LeuCys Leu Ala Gly Arg Gly Leu Gln Glu Ala 1 5 10 136 11 PRT Hepatitis Cvirus 136 Gly Leu Leu Leu Glu Leu Leu Ser Glu His His 1 5 10 137 11 PRTHepatitis C virus 137 Leu Glu Leu Leu Ser Glu His His Pro Leu Leu 1 5 10138 11 PRT Hepatitis C virus 138 Gln Glu Ala Glu Gly Leu Leu Leu Glu LeuLeu 1 5 10 139 11 PRT Hepatitis C virus 139 Glu Gly Leu Leu Leu Glu LeuLeu Ser Glu His 1 5 10 140 11 PRT Hepatitis C virus 140 Gly Arg Gly LeuGln Glu Ala Glu Gly Leu Leu 1 5 10 141 11 PRT Hepatitis C virus 141 AlaGlu Gly Leu Leu Leu Glu Leu Leu Ser Glu 1 5 10 142 11 PRT Hepatitis Cvirus 142 Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu 1 5 10 143 11 PRTHepatitis C virus 143 Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser 1 5 10144 12 PRT Hepatitis C virus 144 Glu Gly Leu Leu Leu Glu Leu Leu Ser GluHis His 1 5 10 145 12 PRT Hepatitis C virus 145 Arg Gly Leu Gln Glu AlaGlu Gly Leu Leu Leu Glu 1 5 10 146 12 PRT Hepatitis C virus 146 Gly LeuLeu Leu Glu Leu Leu Ser Glu His His Pro 1 5 10 147 12 PRT Hepatitis Cvirus 147 Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu 1 5 10 148 12PRT Hepatitis C virus 148 Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu GluLeu 1 5 10 149 12 PRT Hepatitis C virus 149 Leu Ala Gly Arg Gly Leu GlnGlu Ala Glu Gly Leu 1 5 10 150 12 PRT Hepatitis C virus 150 Gly Arg GlyLeu Gln Glu Ala Glu Gly Leu Leu Leu 1 5 10 151 12 PRT Hepatitis C virus151 Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser 1 5 10 152 12 PRTHepatitis C virus 152 Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His 15 10 153 12 PRT Hepatitis C virus 153 Ala Gly Arg Gly Leu Gln Glu AlaGlu Gly Leu Leu 1 5 10 154 12 PRT Hepatitis C virus 154 Leu Glu Leu LeuSer Glu His His Pro Leu Leu Asp 1 5 10 155 12 PRT Hepatitis C virus 155Leu Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu 1 5 10 156 12 PRTHepatitis C virus 156 Leu Leu Leu Glu Leu Leu Ser Glu His His Pro Leu 15 10 157 12 PRT Hepatitis C virus 157 Leu Leu Glu Leu Leu Ser Glu HisHis Pro Leu Leu 1 5 10 158 12 PRT Hepatitis C virus 158 Glu Leu Leu SerGlu His His Pro Leu Leu Asp Val 1 5 10 159 12 PRT Hepatitis C virus 159Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu 1 5 10 160 12 PRTHepatitis C virus 160 Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly 15 10 161 13 PRT Hepatitis C virus 161 Cys Leu Ala Gly Arg Gly Leu GlnGlu Ala Glu Gly Leu 1 5 10 162 13 PRT Hepatitis C virus 162 Arg Gly LeuGln Glu Ala Glu Gly Leu Leu Leu Glu Leu 1 5 10 163 13 PRT Hepatitis Cvirus 163 Leu Leu Glu Leu Leu Ser Glu His His Pro Leu Leu Asp 1 5 10 16413 PRT Hepatitis C virus 164 Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu LeuLeu Glu 1 5 10 165 13 PRT Hepatitis C virus 165 Leu Cys Leu Ala Gly ArgGly Leu Gln Glu Ala Glu Gly 1 5 10 166 13 PRT Hepatitis C virus 166 LeuGlu Leu Leu Ser Glu His His Pro Leu Leu Asp Val 1 5 10 167 13 PRTHepatitis C virus 167 Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His HisPro 1 5 10 168 13 PRT Hepatitis C virus 168 Glu Ala Glu Gly Leu Leu LeuGlu Leu Leu Ser Glu His 1 5 10 169 13 PRT Hepatitis C virus 169 Gly LeuGln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu 1 5 10 170 13 PRT HepatitisC virus 170 Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu 1 5 10171 13 PRT Hepatitis C virus 171 Ala Glu Gly Leu Leu Leu Glu Leu Leu SerGlu His His 1 5 10 172 13 PRT Hepatitis C virus 172 Leu Ala Gly Arg GlyLeu Gln Glu Ala Glu Gly Leu Leu 1 5 10 173 13 PRT Hepatitis C virus 173Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu 1 5 10 174 13 PRTHepatitis C virus 174 Leu Leu Leu Glu Leu Leu Ser Glu His His Pro LeuLeu 1 5 10 175 13 PRT Hepatitis C virus 175 Leu Gln Glu Ala Glu Gly LeuLeu Leu Glu Leu Leu Ser 1 5 10 176 13 PRT Hepatitis C virus 176 Gly LeuLeu Leu Glu Leu Leu Ser Glu His His Pro Leu 1 5 10 177 14 PRT HepatitisC virus 177 Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His His 1 510 178 14 PRT Hepatitis C virus 178 Glu Gly Leu Leu Leu Glu Leu Leu SerGlu His His Pro Leu 1 5 10 179 14 PRT Hepatitis C virus 179 Ala Gly ArgGly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu 1 5 10 180 14 PRT HepatitisC virus 180 Leu Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu 1 510 181 14 PRT Hepatitis C virus 181 Gly Leu Leu Leu Glu Leu Leu Ser GluHis His Pro Leu Leu 1 5 10 182 14 PRT Hepatitis C virus 182 Gln Glu AlaGlu Gly Leu Leu Leu Glu Leu Leu Ser Glu His 1 5 10 183 14 PRT HepatitisC virus 183 Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu 1 510 184 14 PRT Hepatitis C virus 184 Leu Leu Leu Glu Leu Leu Ser Glu HisHis Pro Leu Leu Asp 1 5 10 185 14 PRT Hepatitis C virus 185 Leu Gln GluAla Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu 1 5 10 186 14 PRT HepatitisC virus 186 Leu Leu Glu Leu Leu Ser Glu His His Pro Leu Leu Asp Val 1 510 187 14 PRT Hepatitis C virus 187 Gly Leu Gln Glu Ala Glu Gly Leu LeuLeu Glu Leu Leu Ser 1 5 10 188 14 PRT Hepatitis C virus 188 Gly Arg GlyLeu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu 1 5 10 189 14 PRT HepatitisC virus 189 Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His His Pro 1 510 190 14 PRT Hepatitis C virus 190 Cys Leu Ala Gly Arg Gly Leu Gln GluAla Glu Gly Leu Leu 1 5 10 191 14 PRT Hepatitis C virus 191 Leu Ala GlyArg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu 1 5 10 192 15 PRT HepatitisC virus 192 Leu Leu Leu Glu Leu Leu Ser Glu His His Pro Leu Leu Asp Val1 5 10 15 193 15 PRT Hepatitis C virus 193 Ala Gly Arg Gly Leu Gln GluAla Glu Gly Leu Leu Leu Glu Leu 1 5 10 15 194 15 PRT Hepatitis C virus194 Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His His Pro Leu 1 5 1015 195 15 PRT Hepatitis C virus 195 Gly Leu Leu Leu Glu Leu Leu Ser GluHis His Pro Leu Leu Asp 1 5 10 15 196 15 PRT Hepatitis C virus 196 LeuGln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His 1 5 10 15 197 15PRT Hepatitis C virus 197 Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu GluLeu Leu Ser Glu 1 5 10 15 198 15 PRT Hepatitis C virus 198 Leu Cys LeuAla Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu 1 5 10 15 199 15 PRTHepatitis C virus 199 Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser GluHis His Pro 1 5 10 15 200 15 PRT Hepatitis C virus 200 Gly Arg Gly LeuGln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu 1 5 10 15 201 15 PRTHepatitis C virus 201 Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu SerGlu His His 1 5 10 15 202 15 PRT Hepatitis C virus 202 Leu Ala Gly ArgGly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu 1 5 10 15 203 15 PRTHepatitis C virus 203 Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu GlyLeu Leu Leu 1 5 10 15 204 15 PRT Hepatitis C virus 204 Glu Gly Leu LeuLeu Glu Leu Leu Ser Glu His His Pro Leu Leu 1 5 10 15 205 15 PRTHepatitis C virus 205 Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu GluLeu Leu Ser 1 5 10 15 206 16 PRT Hepatitis C virus 206 Leu Ala Gly ArgGly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu 1 5 10 15 207 16 PRTHepatitis C virus 207 Gly Leu Leu Leu Glu Leu Leu Ser Glu His His ProLeu Leu Asp Val 1 5 10 15 208 16 PRT Hepatitis C virus 208 Gly Arg GlyLeu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser 1 5 10 15 209 16 PRTHepatitis C virus 209 Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu LeuLeu Glu Leu Leu 1 5 10 15 210 16 PRT Hepatitis C virus 210 Gly Leu GlnGlu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His 1 5 10 15 211 16 PRTHepatitis C virus 211 Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu GluLeu Leu Ser Glu 1 5 10 15 212 16 PRT Hepatitis C virus 212 Leu Gln GluAla Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His His 1 5 10 15 213 16 PRTHepatitis C virus 213 Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu GlyLeu Leu Leu Glu 1 5 10 15 214 16 PRT Hepatitis C virus 214 Gln Glu AlaGlu Gly Leu Leu Leu Glu Leu Leu Ser Glu His His Pro 1 5 10 15 215 16 PRTHepatitis C virus 215 Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu HisHis Pro Leu Leu 1 5 10 15 216 16 PRT Hepatitis C virus 216 Glu Ala GluGly Leu Leu Leu Glu Leu Leu Ser Glu His His Pro Leu 1 5 10 15 217 16 PRTHepatitis C virus 217 Leu Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala GluGly Leu Leu Leu 1 5 10 15 218 16 PRT Hepatitis C virus 218 Glu Gly LeuLeu Leu Glu Leu Leu Ser Glu His His Pro Leu Leu Asp 1 5 10 15 219 17 PRTHepatitis C virus 219 Leu Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala GluGly Leu Leu Leu 1 5 10 15 Glu 220 17 PRT Hepatitis C virus 220 Gln GluAla Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His His Pro 1 5 10 15 Leu221 17 PRT Hepatitis C virus 221 Glu Ala Glu Gly Leu Leu Leu Glu Leu LeuSer Glu His His Pro Leu 1 5 10 15 Leu 222 17 PRT Hepatitis C virus 222Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu 1 5 1015 His 223 17 PRT Hepatitis C virus 223 Cys Leu Ala Gly Arg Gly Leu GlnGlu Ala Glu Gly Leu Leu Leu Glu 1 5 10 15 Leu 224 17 PRT Hepatitis Cvirus 224 Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu LeuSer 1 5 10 15 Glu 225 17 PRT Hepatitis C virus 225 Leu Ala Gly Arg GlyLeu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu 1 5 10 15 Leu 226 17 PRTHepatitis C virus 226 Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu LeuLeu Glu Leu Leu 1 5 10 15 Ser 227 17 PRT Hepatitis C virus 227 Gly LeuGln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His 1 5 10 15 His228 17 PRT Hepatitis C virus 228 Glu Gly Leu Leu Leu Glu Leu Leu Ser GluHis His Pro Leu Leu Asp 1 5 10 15 Val 229 17 PRT Hepatitis C virus 229Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His His Pro Leu Leu 1 5 1015 Asp 230 17 PRT Hepatitis C virus 230 Leu Gln Glu Ala Glu Gly Leu LeuLeu Glu Leu Leu Ser Glu His His 1 5 10 15 Pro 231 18 PRT Hepatitis Cvirus 231 Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu LeuGlu 1 5 10 15 Leu Leu 232 18 PRT Hepatitis C virus 232 Leu Gln Glu AlaGlu Gly Leu Leu Leu Glu Leu Leu Ser Glu His His 1 5 10 15 Pro Leu 233 18PRT Hepatitis C virus 233 Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu LeuLeu Glu Leu Leu Ser 1 5 10 15 Glu His 234 18 PRT Hepatitis C virus 234Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His His Pro Leu 1 5 1015 Leu Asp 235 18 PRT Hepatitis C virus 235 Ala Glu Gly Leu Leu Leu GluLeu Leu Ser Glu His His Pro Leu Leu 1 5 10 15 Asp Val 236 18 PRTHepatitis C virus 236 Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu LeuLeu Ser Glu His 1 5 10 15 His Pro 237 18 PRT Hepatitis C virus 237 ArgGly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu 1 5 10 15His His 238 18 PRT Hepatitis C virus 238 Gln Glu Ala Glu Gly Leu Leu LeuGlu Leu Leu Ser Glu His His Pro 1 5 10 15 Leu Leu 239 18 PRT Hepatitis Cvirus 239 Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu GluLeu 1 5 10 15 Leu Ser 240 18 PRT Hepatitis C virus 240 Leu Cys Leu AlaGly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu 1 5 10 15 Glu Leu 241 18PRT Hepatitis C virus 241 Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly LeuLeu Leu Glu Leu Leu 1 5 10 15 Ser Glu 242 19 PRT Hepatitis C virus 242Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu 1 5 1015 Ser Glu His 243 19 PRT Hepatitis C virus 243 Gly Leu Gln Glu Ala GluGly Leu Leu Leu Glu Leu Leu Ser Glu His 1 5 10 15 His Pro Leu 244 19 PRTHepatitis C virus 244 Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu LeuGlu Leu Leu Ser 1 5 10 15 Glu His His 245 19 PRT Hepatitis C virus 245Leu Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu 1 5 1015 Glu Leu Leu 246 19 PRT Hepatitis C virus 246 Cys Leu Ala Gly Arg GlyLeu Gln Glu Ala Glu Gly Leu Leu Leu Glu 1 5 10 15 Leu Leu Ser 247 19 PRTHepatitis C virus 247 Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu GluLeu Leu Ser Glu 1 5 10 15 His His Pro 248 19 PRT Hepatitis C virus 248Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu 1 5 1015 Leu Ser Glu 249 19 PRT Hepatitis C virus 249 Gln Glu Ala Glu Gly LeuLeu Leu Glu Leu Leu Ser Glu His His Pro 1 5 10 15 Leu Leu Asp 250 19 PRTHepatitis C virus 250 Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu LeuSer Glu His His 1 5 10 15 Pro Leu Leu 251 19 PRT Hepatitis C virus 251Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His His Pro Leu 1 5 1015 Leu Asp Val 252 20 PRT Hepatitis C virus 252 Arg Gly Leu Gln Glu AlaGlu Gly Leu Leu Leu Glu Leu Leu Ser Glu 1 5 10 15 His His Pro Leu 20 25320 PRT Hepatitis C virus 253 Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu GluLeu Leu Ser Glu His 1 5 10 15 His Pro Leu Leu 20 254 20 PRT Hepatitis Cvirus 254 Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu LeuLeu 1 5 10 15 Ser Glu His His 20 255 20 PRT Hepatitis C virus 255 LeuAla Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu 1 5 10 15Leu Ser Glu His 20 256 20 PRT Hepatitis C virus 256 Gly Arg Gly Leu GlnGlu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser 1 5 10 15 Glu His His Pro 20257 20 PRT Hepatitis C virus 257 Leu Cys Leu Ala Gly Arg Gly Leu Gln GluAla Glu Gly Leu Leu Leu 1 5 10 15 Glu Leu Leu Ser 20 258 20 PRTHepatitis C virus 258 Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu LeuSer Glu His His 1 5 10 15 Pro Leu Leu Asp 20 259 20 PRT Hepatitis Cvirus 259 Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu Leu Ser Glu His HisPro 1 5 10 15 Leu Leu Asp Val 20 260 20 PRT Hepatitis C virus 260 CysLeu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu 1 5 10 15Leu Leu Ser Glu 20 261 21 PRT Hepatitis C virus 261 Leu Cys Leu Ala GlyArg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu 1 5 10 15 Glu Leu Leu SerGlu 20 262 21 PRT Hepatitis C virus 262 Arg Gly Leu Gln Glu Ala Glu GlyLeu Leu Leu Glu Leu Leu Ser Glu 1 5 10 15 His His Pro Leu Leu 20 263 21PRT Hepatitis C virus 263 Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu LeuLeu Ser Glu His His 1 5 10 15 Pro Leu Leu Asp Val 20 264 21 PRTHepatitis C virus 264 Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu LeuGlu Leu Leu Ser 1 5 10 15 Glu His His Pro Leu 20 265 21 PRT Hepatitis Cvirus 265 Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu LeuLeu 1 5 10 15 Ser Glu His His Pro 20 266 21 PRT Hepatitis C virus 266Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu 1 5 1015 Leu Ser Glu His His 20 267 21 PRT Hepatitis C virus 267 Cys Leu AlaGly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu 1 5 10 15 Leu LeuSer Glu His 20 268 21 PRT Hepatitis C virus 268 Gly Leu Gln Glu Ala GluGly Leu Leu Leu Glu Leu Leu Ser Glu His 1 5 10 15 His Pro Leu Leu Asp 20269 22 PRT Hepatitis C virus 269 Gly Leu Gln Glu Ala Glu Gly Leu Leu LeuGlu Leu Leu Ser Glu His 1 5 10 15 His Pro Leu Leu Asp Val 20 270 22 PRTHepatitis C virus 270 Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu LeuLeu Glu Leu Leu 1 5 10 15 Ser Glu His His Pro Leu 20 271 22 PRTHepatitis C virus 271 Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu GlyLeu Leu Leu Glu 1 5 10 15 Leu Leu Ser Glu His His 20 272 22 PRTHepatitis C virus 272 Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu LeuGlu Leu Leu Ser 1 5 10 15 Glu His His Pro Leu Leu 20 273 22 PRTHepatitis C virus 273 Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu GluLeu Leu Ser Glu 1 5 10 15 His His Pro Leu Leu Asp 20 274 22 PRTHepatitis C virus 274 Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly LeuLeu Leu Glu Leu 1 5 10 15 Leu Ser Glu His His Pro 20 275 22 PRTHepatitis C virus 275 Leu Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala GluGly Leu Leu Leu 1 5 10 15 Glu Leu Leu Ser Glu His 20 276 23 PRTHepatitis C virus 276 Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu GlyLeu Leu Leu Glu 1 5 10 15 Leu Leu Ser Glu His His Pro 20 277 23 PRTHepatitis C virus 277 Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu LeuGlu Leu Leu Ser 1 5 10 15 Glu His His Pro Leu Leu Asp 20 278 23 PRTHepatitis C virus 278 Leu Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala GluGly Leu Leu Leu 1 5 10 15 Glu Leu Leu Ser Glu His His 20 279 23 PRTHepatitis C virus 279 Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly LeuLeu Leu Glu Leu 1 5 10 15 Leu Ser Glu His His Pro Leu 20 280 23 PRTHepatitis C virus 280 Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu GluLeu Leu Ser Glu 1 5 10 15 His His Pro Leu Leu Asp Val 20 281 23 PRTHepatitis C virus 281 Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu LeuLeu Glu Leu Leu 1 5 10 15 Ser Glu His His Pro Leu Leu 20 282 24 PRTHepatitis C virus 282 Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu LeuLeu Glu Leu Leu 1 5 10 15 Ser Glu His His Pro Leu Leu Asp 20 283 24 PRTHepatitis C virus 283 Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu GlyLeu Leu Leu Glu 1 5 10 15 Leu Leu Ser Glu His His Pro Leu 20 284 24 PRTHepatitis C virus 284 Leu Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala GluGly Leu Leu Leu 1 5 10 15 Glu Leu Leu Ser Glu His His Pro 20 285 24 PRTHepatitis C virus 285 Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu LeuGlu Leu Leu Ser 1 5 10 15 Glu His His Pro Leu Leu Asp Val 20 286 24 PRTHepatitis C virus 286 Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly LeuLeu Leu Glu Leu 1 5 10 15 Leu Ser Glu His His Pro Leu Leu 20 287 25 PRTHepatitis C virus 287 Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu LeuLeu Glu Leu Leu 1 5 10 15 Ser Glu His His Pro Leu Leu Asp Val 20 25 28825 PRT Hepatitis C virus 288 Leu Cys Leu Ala Gly Arg Gly Leu Gln Glu AlaGlu Gly Leu Leu Leu 1 5 10 15 Glu Leu Leu Ser Glu His His Pro Leu 20 25289 25 PRT Hepatitis C virus 289 Leu Ala Gly Arg Gly Leu Gln Glu Ala GluGly Leu Leu Leu Glu Leu 1 5 10 15 Leu Ser Glu His His Pro Leu Leu Asp 2025 290 25 PRT Hepatitis C virus 290 Cys Leu Ala Gly Arg Gly Leu Gln GluAla Glu Gly Leu Leu Leu Glu 1 5 10 15 Leu Leu Ser Glu His His Pro LeuLeu 20 25 291 26 PRT Hepatitis C virus 291 Leu Cys Leu Ala Gly Arg GlyLeu Gln Glu Ala Glu Gly Leu Leu Leu 1 5 10 15 Glu Leu Leu Ser Glu HisHis Pro Leu Leu 20 25 292 26 PRT Hepatitis C virus 292 Leu Ala Gly ArgGly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu Leu 1 5 10 15 Leu Ser GluHis His Pro Leu Leu Asp Val 20 25 293 26 PRT Hepatitis C virus 293 CysLeu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu Leu Leu Glu 1 5 10 15Leu Leu Ser Glu His His Pro Leu Leu Asp 20 25 294 27 PRT Hepatitis Cvirus 294 Leu Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala Glu Gly Leu LeuLeu 1 5 10 15 Glu Leu Leu Ser Glu His His Pro Leu Leu Asp 20 25 295 27PRT Hepatitis C virus 295 Cys Leu Ala Gly Arg Gly Leu Gln Glu Ala GluGly Leu Leu Leu Glu 1 5 10 15 Leu Leu Ser Glu His His Pro Leu Leu AspVal 20 25 296 72 RNA Hepatitis C virus 296 uggcuaaggg acaucugggacuggauaugc gaggugcuga gcgacuuuaa gaccuggcug 60 aaagccaagc uc 72 297 24PRT Hepatitis C virus 297 Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys GluVal Leu Ser Asp Phe 1 5 10 15 Lys Thr Trp Leu Lys Ala Lys Leu 20 298 72DNA Hepatitis C virus 298 gagcttggct ttcagccagg tcttaaagtc gctcagcacctcgcatatcc agtcccagat 60 gtcccttagc ca 72 299 22 PRT Hepatitis C virus299 Glu Leu Gly Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu Ala Tyr 1 510 15 Pro Val Pro Asp Val Pro 20 300 6 PRT Hepatitis C virus 300 Ala GlnHis Leu Ala Tyr 1 5 301 6 PRT Hepatitis C virus 301 Leu Gly Phe Gln ProGly 1 5 302 6 PRT Hepatitis C virus 302 Gly Leu Lys Val Ala Gln 1 5 3036 PRT Hepatitis C virus 303 Glu Leu Gly Phe Gln Pro 1 5 304 6 PRTHepatitis C virus 304 Pro Val Pro Asp Val Pro 1 5 305 6 PRT Hepatitis Cvirus 305 Phe Gln Pro Gly Leu Lys 1 5 306 6 PRT Hepatitis C virus 306Gly Phe Gln Pro Gly Leu 1 5 307 6 PRT Hepatitis C virus 307 Val Ala GlnHis Leu Ala 1 5 308 6 PRT Hepatitis C virus 308 Leu Lys Val Ala Gln His1 5 309 6 PRT Hepatitis C virus 309 His Leu Ala Tyr Pro Val 1 5 310 6PRT Hepatitis C virus 310 Lys Val Ala Gln His Leu 1 5 311 6 PRTHepatitis C virus 311 Gln Pro Gly Leu Lys Val 1 5 312 6 PRT Hepatitis Cvirus 312 Leu Ala Tyr Pro Val Pro 1 5 313 6 PRT Hepatitis C virus 313Tyr Pro Val Pro Asp Val 1 5 314 6 PRT Hepatitis C virus 314 Gln His LeuAla Tyr Pro 1 5 315 6 PRT Hepatitis C virus 315 Pro Gly Leu Lys Val Ala1 5 316 6 PRT Hepatitis C virus 316 Ala Tyr Pro Val Pro Asp 1 5 317 7PRT Hepatitis C virus 317 Gln Pro Gly Leu Lys Val Ala 1 5 318 7 PRTHepatitis C virus 318 Glu Leu Gly Phe Gln Pro Gly 1 5 319 7 PRTHepatitis C virus 319 His Leu Ala Tyr Pro Val Pro 1 5 320 7 PRTHepatitis C virus 320 Leu Ala Tyr Pro Val Pro Asp 1 5 321 7 PRTHepatitis C virus 321 Ala Gln His Leu Ala Tyr Pro 1 5 322 7 PRTHepatitis C virus 322 Phe Gln Pro Gly Leu Lys Val 1 5 323 7 PRTHepatitis C virus 323 Lys Val Ala Gln His Leu Ala 1 5 324 7 PRTHepatitis C virus 324 Leu Gly Phe Gln Pro Gly Leu 1 5 325 7 PRTHepatitis C virus 325 Ala Tyr Pro Val Pro Asp Val 1 5 326 7 PRTHepatitis C virus 326 Pro Gly Leu Lys Val Ala Gln 1 5 327 7 PRTHepatitis C virus 327 Leu Lys Val Ala Gln His Leu 1 5 328 7 PRTHepatitis C virus 328 Gly Leu Lys Val Ala Gln His 1 5 329 7 PRTHepatitis C virus 329 Val Ala Gln His Leu Ala Tyr 1 5 330 7 PRTHepatitis C virus 330 Gly Phe Gln Pro Gly Leu Lys 1 5 331 7 PRTHepatitis C virus 331 Gln His Leu Ala Tyr Pro Val 1 5 332 7 PRTHepatitis C virus 332 Tyr Pro Val Pro Asp Val Pro 1 5 333 8 PRTHepatitis C virus 333 Ala Tyr Pro Val Pro Asp Val Pro 1 5 334 8 PRTHepatitis C virus 334 Pro Gly Leu Lys Val Ala Gln His 1 5 335 8 PRTHepatitis C virus 335 Gln His Leu Ala Tyr Pro Val Pro 1 5 336 8 PRTHepatitis C virus 336 Leu Ala Tyr Pro Val Pro Asp Val 1 5 337 8 PRTHepatitis C virus 337 Leu Lys Val Ala Gln His Leu Ala 1 5 338 8 PRTHepatitis C virus 338 Gly Leu Lys Val Ala Gln His Leu 1 5 339 8 PRTHepatitis C virus 339 Gly Phe Gln Pro Gly Leu Lys Val 1 5 340 8 PRTHepatitis C virus 340 Ala Gln His Leu Ala Tyr Pro Val 1 5 341 8 PRTHepatitis C virus 341 Val Ala Gln His Leu Ala Tyr Pro 1 5 342 8 PRTHepatitis C virus 342 Lys Val Ala Gln His Leu Ala Tyr 1 5 343 8 PRTHepatitis C virus 343 Glu Leu Gly Phe Gln Pro Gly Leu 1 5 344 8 PRTHepatitis C virus 344 Leu Gly Phe Gln Pro Gly Leu Lys 1 5 345 8 PRTHepatitis C virus 345 Phe Gln Pro Gly Leu Lys Val Ala 1 5 346 8 PRTHepatitis C virus 346 His Leu Ala Tyr Pro Val Pro Asp 1 5 347 8 PRTHepatitis C virus 347 Gln Pro Gly Leu Lys Val Ala Gln 1 5 348 9 PRTHepatitis C virus 348 Glu Leu Gly Phe Gln Pro Gly Leu Lys 1 5 349 9 PRTHepatitis C virus 349 Val Ala Gln His Leu Ala Tyr Pro Val 1 5 350 9 PRTHepatitis C virus 350 Leu Gly Phe Gln Pro Gly Leu Lys Val 1 5 351 9 PRTHepatitis C virus 351 Gln Pro Gly Leu Lys Val Ala Gln His 1 5 352 9 PRTHepatitis C virus 352 Lys Val Ala Gln His Leu Ala Tyr Pro 1 5 353 9 PRTHepatitis C virus 353 Pro Gly Leu Lys Val Ala Gln His Leu 1 5 354 9 PRTHepatitis C virus 354 Phe Gln Pro Gly Leu Lys Val Ala Gln 1 5 355 9 PRTHepatitis C virus 355 Gly Leu Lys Val Ala Gln His Leu Ala 1 5 356 9 PRTHepatitis C virus 356 Leu Ala Tyr Pro Val Pro Asp Val Pro 1 5 357 9 PRTHepatitis C virus 357 His Leu Ala Tyr Pro Val Pro Asp Val 1 5 358 9 PRTHepatitis C virus 358 Gly Phe Gln Pro Gly Leu Lys Val Ala 1 5 359 9 PRTHepatitis C virus 359 Ala Gln His Leu Ala Tyr Pro Val Pro 1 5 360 9 PRTHepatitis C virus 360 Gln His Leu Ala Tyr Pro Val Pro Asp 1 5 361 9 PRTHepatitis C virus 361 Leu Lys Val Ala Gln His Leu Ala Tyr 1 5 362 10 PRTHepatitis C virus 362 Gln Pro Gly Leu Lys Val Ala Gln His Leu 1 5 10 36310 PRT Hepatitis C virus 363 Pro Gly Leu Lys Val Ala Gln His Leu Ala 1 510 364 10 PRT Hepatitis C virus 364 His Leu Ala Tyr Pro Val Pro Asp ValPro 1 5 10 365 10 PRT Hepatitis C virus 365 Gly Leu Lys Val Ala Gln HisLeu Ala Tyr 1 5 10 366 10 PRT Hepatitis C virus 366 Val Ala Gln His LeuAla Tyr Pro Val Pro 1 5 10 367 10 PRT Hepatitis C virus 367 Lys Val AlaGln His Leu Ala Tyr Pro Val 1 5 10 368 10 PRT Hepatitis C virus 368 GlyPhe Gln Pro Gly Leu Lys Val Ala Gln 1 5 10 369 10 PRT Hepatitis C virus369 Gln His Leu Ala Tyr Pro Val Pro Asp Val 1 5 10 370 10 PRT HepatitisC virus 370 Ala Gln His Leu Ala Tyr Pro Val Pro Asp 1 5 10 371 10 PRTHepatitis C virus 371 Phe Gln Pro Gly Leu Lys Val Ala Gln His 1 5 10 37210 PRT Hepatitis C virus 372 Glu Leu Gly Phe Gln Pro Gly Leu Lys Val 1 510 373 10 PRT Hepatitis C virus 373 Leu Lys Val Ala Gln His Leu Ala TyrPro 1 5 10 374 10 PRT Hepatitis C virus 374 Leu Gly Phe Gln Pro Gly LeuLys Val Ala 1 5 10 375 11 PRT Hepatitis C virus 375 Gln His Leu Ala TyrPro Val Pro Asp Val Pro 1 5 10 376 11 PRT Hepatitis C virus 376 Leu LysVal Ala Gln His Leu Ala Tyr Pro Val 1 5 10 377 11 PRT Hepatitis C virus377 Val Ala Gln His Leu Ala Tyr Pro Val Pro Asp 1 5 10 378 11 PRTHepatitis C virus 378 Pro Gly Leu Lys Val Ala Gln His Leu Ala Tyr 1 5 10379 11 PRT Hepatitis C virus 379 Leu Gly Phe Gln Pro Gly Leu Lys Val AlaGln 1 5 10 380 11 PRT Hepatitis C virus 380 Lys Val Ala Gln His Leu AlaTyr Pro Val Pro 1 5 10 381 11 PRT Hepatitis C virus 381 Glu Leu Gly PheGln Pro Gly Leu Lys Val Ala 1 5 10 382 11 PRT Hepatitis C virus 382 GlyLeu Lys Val Ala Gln His Leu Ala Tyr Pro 1 5 10 383 11 PRT Hepatitis Cvirus 383 Ala Gln His Leu Ala Tyr Pro Val Pro Asp Val 1 5 10 384 11 PRTHepatitis C virus 384 Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu 1 5 10385 11 PRT Hepatitis C virus 385 Gln Pro Gly Leu Lys Val Ala Gln His LeuAla 1 5 10 386 11 PRT Hepatitis C virus 386 Gly Phe Gln Pro Gly Leu LysVal Ala Gln His 1 5 10 387 12 PRT Hepatitis C virus 387 Leu Gly Phe GlnPro Gly Leu Lys Val Ala Gln His 1 5 10 388 12 PRT Hepatitis C virus 388Pro Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro 1 5 10 389 12 PRTHepatitis C virus 389 Leu Lys Val Ala Gln His Leu Ala Tyr Pro Val Pro 15 10 390 12 PRT Hepatitis C virus 390 Gly Phe Gln Pro Gly Leu Lys ValAla Gln His Leu 1 5 10 391 12 PRT Hepatitis C virus 391 Val Ala Gln HisLeu Ala Tyr Pro Val Pro Asp Val 1 5 10 392 12 PRT Hepatitis C virus 392Gln Pro Gly Leu Lys Val Ala Gln His Leu Ala Tyr 1 5 10 393 12 PRTHepatitis C virus 393 Glu Leu Gly Phe Gln Pro Gly Leu Lys Val Ala Gln 15 10 394 12 PRT Hepatitis C virus 394 Ala Gln His Leu Ala Tyr Pro ValPro Asp Val Pro 1 5 10 395 12 PRT Hepatitis C virus 395 Lys Val Ala GlnHis Leu Ala Tyr Pro Val Pro Asp 1 5 10 396 12 PRT Hepatitis C virus 396Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu Ala 1 5 10 397 12 PRTHepatitis C virus 397 Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro Val 15 10 398 13 PRT Hepatitis C virus 398 Leu Gly Phe Gln Pro Gly Leu LysVal Ala Gln His Leu 1 5 10 399 13 PRT Hepatitis C virus 399 Val Ala GlnHis Leu Ala Tyr Pro Val Pro Asp Val Pro 1 5 10 400 13 PRT Hepatitis Cvirus 400 Pro Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro Val 1 5 10 40113 PRT Hepatitis C virus 401 Gly Phe Gln Pro Gly Leu Lys Val Ala Gln HisLeu Ala 1 5 10 402 13 PRT Hepatitis C virus 402 Leu Lys Val Ala Gln HisLeu Ala Tyr Pro Val Pro Asp 1 5 10 403 13 PRT Hepatitis C virus 403 GlnPro Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro 1 5 10 404 13 PRTHepatitis C virus 404 Glu Leu Gly Phe Gln Pro Gly Leu Lys Val Ala GlnHis 1 5 10 405 13 PRT Hepatitis C virus 405 Gly Leu Lys Val Ala Gln HisLeu Ala Tyr Pro Val Pro 1 5 10 406 13 PRT Hepatitis C virus 406 Phe GlnPro Gly Leu Lys Val Ala Gln His Leu Ala Tyr 1 5 10 407 13 PRT HepatitisC virus 407 Lys Val Ala Gln His Leu Ala Tyr Pro Val Pro Asp Val 1 5 10408 14 PRT Hepatitis C virus 408 Glu Leu Gly Phe Gln Pro Gly Leu Lys ValAla Gln His Leu 1 5 10 409 14 PRT Hepatitis C virus 409 Gln Pro Gly LeuLys Val Ala Gln His Leu Ala Tyr Pro Val 1 5 10 410 14 PRT Hepatitis Cvirus 410 Gly Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu Ala Tyr 1 5 10411 14 PRT Hepatitis C virus 411 Leu Gly Phe Gln Pro Gly Leu Lys Val AlaGln His Leu Ala 1 5 10 412 14 PRT Hepatitis C virus 412 Phe Gln Pro GlyLeu Lys Val Ala Gln His Leu Ala Tyr Pro 1 5 10 413 14 PRT Hepatitis Cvirus 413 Lys Val Ala Gln His Leu Ala Tyr Pro Val Pro Asp Val Pro 1 5 10414 14 PRT Hepatitis C virus 414 Pro Gly Leu Lys Val Ala Gln His Leu AlaTyr Pro Val Pro 1 5 10 415 14 PRT Hepatitis C virus 415 Leu Lys Val AlaGln His Leu Ala Tyr Pro Val Pro Asp Val 1 5 10 416 14 PRT Hepatitis Cvirus 416 Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro Val Pro Asp 1 5 10417 15 PRT Hepatitis C virus 417 Gly Leu Lys Val Ala Gln His Leu Ala TyrPro Val Pro Asp Val 1 5 10 15 418 15 PRT Hepatitis C virus 418 Pro GlyLeu Lys Val Ala Gln His Leu Ala Tyr Pro Val Pro Asp 1 5 10 15 419 15 PRTHepatitis C virus 419 Glu Leu Gly Phe Gln Pro Gly Leu Lys Val Ala GlnHis Leu Ala 1 5 10 15 420 15 PRT Hepatitis C virus 420 Leu Gly Phe GlnPro Gly Leu Lys Val Ala Gln His Leu Ala Tyr 1 5 10 15 421 15 PRTHepatitis C virus 421 Gly Phe Gln Pro Gly Leu Lys Val Ala Gln His LeuAla Tyr Pro 1 5 10 15 422 15 PRT Hepatitis C virus 422 Gln Pro Gly LeuLys Val Ala Gln His Leu Ala Tyr Pro Val Pro 1 5 10 15 423 15 PRTHepatitis C virus 423 Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu AlaTyr Pro Val 1 5 10 15 424 15 PRT Hepatitis C virus 424 Leu Lys Val AlaGln His Leu Ala Tyr Pro Val Pro Asp Val Pro 1 5 10 15 425 16 PRTHepatitis C virus 425 Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu AlaTyr Pro Val Pro 1 5 10 15 426 16 PRT Hepatitis C virus 426 Gly Phe GlnPro Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro Val 1 5 10 15 427 16 PRTHepatitis C virus 427 Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro ValPro Asp Val Pro 1 5 10 15 428 16 PRT Hepatitis C virus 428 Pro Gly LeuLys Val Ala Gln His Leu Ala Tyr Pro Val Pro Asp Val 1 5 10 15 429 16 PRTHepatitis C virus 429 Gln Pro Gly Leu Lys Val Ala Gln His Leu Ala TyrPro Val Pro Asp 1 5 10 15 430 16 PRT Hepatitis C virus 430 Glu Leu GlyPhe Gln Pro Gly Leu Lys Val Ala Gln His Leu Ala Tyr 1 5 10 15 431 16 PRTHepatitis C virus 431 Leu Gly Phe Gln Pro Gly Leu Lys Val Ala Gln HisLeu Ala Tyr Pro 1 5 10 15 432 17 PRT Hepatitis C virus 432 Gly Phe GlnPro Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro Val 1 5 10 15 Pro 433 17PRT Hepatitis C virus 433 Phe Gln Pro Gly Leu Lys Val Ala Gln His LeuAla Tyr Pro Val Pro 1 5 10 15 Asp 434 17 PRT Hepatitis C virus 434 GlnPro Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro Val Pro Asp 1 5 10 15Val 435 17 PRT Hepatitis C virus 435 Pro Gly Leu Lys Val Ala Gln His LeuAla Tyr Pro Val Pro Asp Val 1 5 10 15 Pro 436 17 PRT Hepatitis C virus436 Leu Gly Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro 1 510 15 Val 437 17 PRT Hepatitis C virus 437 Glu Leu Gly Phe Gln Pro GlyLeu Lys Val Ala Gln His Leu Ala Tyr 1 5 10 15 Pro 438 18 PRT Hepatitis Cvirus 438 Glu Leu Gly Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu AlaTyr 1 5 10 15 Pro Val 439 18 PRT Hepatitis C virus 439 Gln Pro Gly LeuLys Val Ala Gln His Leu Ala Tyr Pro Val Pro Asp 1 5 10 15 Val Pro 440 18PRT Hepatitis C virus 440 Leu Gly Phe Gln Pro Gly Leu Lys Val Ala GlnHis Leu Ala Tyr Pro 1 5 10 15 Val Pro 441 18 PRT Hepatitis C virus 441Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro Val Pro 1 5 1015 Asp Val 442 18 PRT Hepatitis C virus 442 Gly Phe Gln Pro Gly Leu LysVal Ala Gln His Leu Ala Tyr Pro Val 1 5 10 15 Pro Asp 443 19 PRTHepatitis C virus 443 Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu AlaTyr Pro Val Pro 1 5 10 15 Asp Val Pro 444 19 PRT Hepatitis C virus 444Leu Gly Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro 1 5 1015 Val Pro Asp 445 19 PRT Hepatitis C virus 445 Gly Phe Gln Pro Gly LeuLys Val Ala Gln His Leu Ala Tyr Pro Val 1 5 10 15 Pro Asp Val 446 19 PRTHepatitis C virus 446 Glu Leu Gly Phe Gln Pro Gly Leu Lys Val Ala GlnHis Leu Ala Tyr 1 5 10 15 Pro Val Pro 447 20 PRT Hepatitis C virus 447Leu Gly Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu Ala Tyr Pro 1 5 1015 Val Pro Asp Val 20 448 20 PRT Hepatitis C virus 448 Glu Leu Gly PheGln Pro Gly Leu Lys Val Ala Gln His Leu Ala Tyr 1 5 10 15 Pro Val ProAsp 20 449 20 PRT Hepatitis C virus 449 Gly Phe Gln Pro Gly Leu Lys ValAla Gln His Leu Ala Tyr Pro Val 1 5 10 15 Pro Asp Val Pro 20 450 21 PRTHepatitis C virus 450 Glu Leu Gly Phe Gln Pro Gly Leu Lys Val Ala GlnHis Leu Ala Tyr 1 5 10 15 Pro Val Pro Asp Val 20 451 21 PRT Hepatitis Cvirus 451 Leu Gly Phe Gln Pro Gly Leu Lys Val Ala Gln His Leu Ala TyrPro 1 5 10 15 Val Pro Asp Val Pro 20 452 63 DNA Artificial sequence PCRprimer 452 tccggctcct ggctaaggga cgactgggac tgggaatgcg aggtgctgagcgacgataag 60 acc 63 453 45 DNA Artificial sequence PCR primer 453gattgggatt gggaatgcac ggtgttgact gatgacaaga cctgg 45 454 13 PRTHepatitis C virus 454 His Asp Ser Phe Ala Asn Ala Thr Gly Arg Phe TrpPro 1 5 10 455 13 PRT Hepatitis C virus 455 Gln Gly Thr Ser Pro Ser ArgLeu Ala Val Pro Leu Ala 1 5 10 456 13 PRT Hepatitis C virus 456 Ile SerSer Lys Thr Gly Met Ser Ser Glu Pro Pro Ser 1 5 10 457 13 PRT HepatitisC virus 457 Ile Leu Ser Ser Ile Asp Ala Leu Gly Ser Asp Ser His 1 5 10458 13 PRT Hepatitis C virus 458 Leu Asp Asp Arg Ser Val Pro Thr Val IleSer Gln Arg 1 5 10 459 13 PRT Hepatitis C virus 459 Tyr Pro Ser Lys ProGly Asn Val Thr Pro Lys Ala Pro 1 5 10 460 9 PRT Hepatitis C virus 460Gln Ala Gln Gly Glu Arg Ala Leu Lys 1 5 461 13 PRT Hepatitis C virus 461Thr Asp Lys Arg Ala Ser Pro Leu Thr Val Gln Ala Arg 1 5 10 462 29 PRTHepatitis C virus 462 Tyr Ile Glu Gln Gly Met Met Leu Ala Glu Gln PheLys Gln Lys Ala 1 5 10 15 Leu Gly Leu Leu Gln Thr Ala Ser Arg His AlaGlu Val 20 25 463 17 PRT Artificial sequence Mutated version ofnon-structural protein NS5A 463 Leu Arg Asp Val Trp Asp Trp Ile Cys ThrVal Leu Thr Asp Phe Lys 1 5 10 15 Thr 464 10 PRT Artificial sequenceMutated version of non-structural protein NS5A 464 Leu Arg Asp Val TrpAsp Trp Ile Cys Thr 1 5 10 465 12 PRT Artificial sequence Mutatedversion of non-structural protein NS5A 465 Asp Val Trp Asp Trp Ile CysThr Val Leu Thr Asp 1 5 10 466 11 PRT Artificial sequence Mutatedversion of non-structural protein NS5A 466 Ser Trp Leu Arg Asp Val TrpAsp Trp Ile Cys 1 5 10 467 27 PRT Artificial sequence Mutated version ofnon-structural protein NS5A 467 Ser Gly Ser Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln SerLys Leu 20 25 468 17 PRT Artificial sequence Mutated version ofnon-structural protein NS5A 468 Xaa Xaa Asp Val Trp Asp Trp Ile Cys ThrXaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa 469 24 PRT Hepatitis C virus 469Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu Val Leu Cys Asp Phe 1 5 1015 Lys Thr Trp Leu Lys Ala Lys Leu 20 470 24 PRT Hepatitis C virus 470Trp Leu Arg Asp Asp Trp Asp Trp Glu Cys Glu Val Leu Ser Asp Asp 1 5 1015 Lys Thr Trp Leu Lys Ala Lys Leu 20

What is claimed is:
 1. A method to identify a compound useful to treatHepatitis C virus (HCV) infection comprising assessing the ability of acandidate compound to interfere with the binding of an amphipathic helixpresent in the N-terminal region of an HCV nonstructural protein withcytoplasmic membranes of a eukaryotic cell, where a compound whichinterferes with the binding is identified as being useful in treatingHCV infection.
 2. The method of claim 1, wherein the assessing isperformed extracellularly.
 3. The method of claim 2, wherein theassessing comprises determining the ability of a compound to bind to theamphipathic helix, wherein a compound which binds to the amphipathichelix with high affinity is identified as a compound useful in treatingHCV infection.
 4. The method of claim 2, wherein the assessing comprisesdetermining the binding of the amphipathic helix to a cell membranepreparation in the presence of a candidate compound and in the absenceof a candidate compound, wherein a decrease in the binding of theamphipathic helix to the cell membrane preparation in the presence ofthe compound as compared to binding in the absence of the compoundidentifies the compound as being useful in treating HCV infection. 5.The method of claim 1, wherein the method is performed intracellularlyin a eukaryotic cell.
 6. The method of claim 5, wherein the amphipathichelix is coupled to a detectable label and the intracellulardistribution of the label is assessed in the presence of a candidatecompound, wherein the candidate compound is identified as being usefulin the treatment of HCV infection when its presence results in anintracellular distribution of the label which does not show binding tocytoplasmic membranes in comparison to the distribution of the label inthe absence of the compound.
 7. The method of claim 6, wherein thedetectable label is green fluorescent protein.
 8. A compound useful totreat HCV infection identified by the method of any one of claims 1 to7.
 9. A method to treat HCV infection comprising administering to asubject in need of such treatment an anti-HCV effective amount of acompound identified by the method of any one of claims 1 to
 7. 10. Amethod to treat HCV infection in a subject comprising administering to asubject in need of such treatment an anti-HCV effective amount of atleast one compound which inhibits the binding of an amphipathic helixpresent in the N-terminal region of an HCV nonstructural protein withcytoplasmic membranes.
 11. The method of claim 10, wherein the at leastone compound is a compound that interacts with one or more sites on thecytoplasmic membrane to which the amphipathic helix binds.
 12. Themethod of claim 11, wherein the at least one compound is a compoundwhich mimics the charge/shape configuration of an amphipathic helixpresent in the N-terminal region of an HCV nonstructural protein. 13.The method of claim 12, wherein the compound comprises the amphipathichelix present in the N-terminal region of an HCV nonstructural proteinor is a peptide having at least about 80% sequence identity with theamphipathic helix.
 14. The method of claim 10, wherein at least onecompound interacts with the amphipathic helix.
 15. A composition foreliciting an immunological response against HCV in a subject comprisingan HCV replicon, wherein the nucleotide sequence encoding an amphipathichelix in the N-terminal region of an HCV nonstructural protein isdeleted or altered to disrupt the amphipathic nature of the amphipathichelix.
 16. The composition of claim 15, wherein the HCV nonstructuralprotein is NS5A, NS5B or NS4B.
 17. The composition of claim 16, whereinthe HCV non-structural protein is NS5A.
 18. A method to elicit animmunological response against HCV in a subject comprising administeringto the subject an anti-HCV effective amount of the composition of claim17 sufficient to elicit the response.
 19. An isolated peptide of fromabout 4 to 60 amino acid residues, which comprises an amphipathic helixpresent in the N-terminal region of an HCV nonstructural protein, thepeptide optionally comprising non-peptide isosteric linkages, whereinthe peptide is optionally coupled to an additional heterologous secondpeptide or to an additional component.
 20. The peptide of claim 19,which is coupled to a membrane-penetrating facilitator.
 21. The methodof claim 20, wherein the membrane-penetrating facilitator is a membranetransport sequence or a TAT peptide.
 22. The peptide of claim 19, whichis coupled to a detectable label.
 23. An isolated peptide consistingessentially of an amino acid sequence selected from the group consistingof SEQ ID NOS: 1 to 13 and
 462. 24. The peptide of claim 23, which iscoupled to a membrane-penetrating facilitator.
 25. The peptide of claim23, which is coupled to a detectable label.
 26. The compound of claim 8,which is a peptide having the following amino acid sequence:SGSWLRDVWDWICTVLTDFKTWLQSKL. (SEQ ID NO: 14)


27. The peptide of claim 26, wherein the peptide comprises one or moreof the following amino acid substitutions: substitution of L at aminoacid position 16 by A or K; or substitution of T at amino acid position17 by A; or substitution of D at amino acid position 18 by A; orsubstitution of F at amino acid position 19 by A; or substitution of Kat amino acid position 20 by A; or substitution of W at amino acidposition 22 by A; or substitution of L at amino acid position 23 by K.28. The peptide of claim 26 or 27, wherein the peptide inhibits bindingof HCV nonstructural proteins with a cytoplasmic membrane of aeukaryotic cell.
 29. The peptide of claim 26 or 27, wherein the HCVnonstructural protein is NS5A.
 30. The compound of claim 8, which is apeptide having the following amino acid sequence:SGSX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈ X₁₉X₂₀QSKL wherein X₁is A or F; X₂ is A or K; X₃ is A or N; X₄ is A or S; X₅ is D, I or S; X₆is A or F; X₇ is A or S; X₈ is A or F; X₉ is E or L; X₁₀ is A or S; X₁₁is A, E or W; X₁₂ is D or S; X₁₃ is A or K; X₁₄ is A, S or W; X₁₅ is Aor S; X₁₆ is D or L; X₁₇ is A or L; X₁₈ is A or W; X₁₉ is A or F; andX₂₀ is A or K.


31. The compound of claim 30, wherein the peptide inhibits binding ofHCV nonstructural proteins with the cytoplasmic membrane.
 32. Thecompound of claim 31, wherein the HCV nonstructural protein is NS5A. 33.An isolated peptide having the following amino acid sequence:SGSWLRDVWDWICTVLTDFKTWLQSKL. (SEQ ID NO: 14)


34. The compound of claim 33, wherein the peptide comprises one or moreof the following amino acid substitutions: substitution of L at aminoacid position 16 by A or K; or substitution of T at amino acid position17 by A; or substitution of D at amino acid position 18 by A; orsubstitution of F at amino acid position 19 by A; or substitution of Kat amino acid position 20 by A; or substitution of W at amino acidposition 22 by A; or substitution of L at amino acid position 23 by K.35. An isolated peptide having the following amino acid sequence:X₁X₂DVWDWICTX₃X₄X₅X₆X₇X₈X₉,

with the provisos that when X₁ and X₂ are present, X₁ is L and X₂ is R;wherein when X₁ and X₂ are present, X₃, X₄, X₅, X₆, X₇, X₈ and X₉ areoptionally present, and when X₃, X₄, X₅, X₆, X₇, X₈ and X₉ are present,X₃ is V, X₄ is L, X₅ is T, X₆ is D, X₇ is F, X₈ is K and X₉ is T; andwherein when X₆ is present, X₃, X₄ and X₅ are all present, and X₆ is D,X₃ is V, X₄is L, and X₅ is T.
 36. The peptide of claim 35, having theamino acid sequence: LRDVWDWICTVLTDFKT, (SEQ ID NO: 463) LRDVWDWICT (SEQID NO: 464) or DVWDWICTVLTD. (SEQ ID NO: 465)


37. An isolated peptide having the amino acid sequence: SWLRDVWDWIC.(SEQ ID NO: 466)


38. An isolated peptide having the amino acid sequence:LCLAGRGLQEAEGLLLELLSEHHPLLDV (SEQ ID NO: 20) or any functional fragmentthereof.
 39. The isolated peptide of claim 38, wherein the functionalfragment is 6 to 27 amino acids in length.
 40. An isolated peptidehaving the amino acid sequence ELGFQPGLKVAQHLAYPVPDVP (SEQ ID NO: 299)or any functional fragment thereof
 41. The isolated peptide of claim 40,wherein the functional fragment is 6 to 21 amino acids in length.
 42. Amethod of identifying a peptide that inhibits the binding of an HCVnonstructural protein to a cytoplasmic membrane of a eukaryotic cell,comprising contacting an amphipathic helix obtained from the N-terminalregion of an HCV nonstructural protein with cytoplasmic membranes of aeukaryotic cell in the presence and absence of a test peptide, wherein adecreased level of binding of the amphipathic helix to the cytoplasmicmembrane in the presence of the test peptide as compared to the level ofbinding of the amphipathic helix to the cytoplasmic membrane in theabsence of the test peptide identifies the test peptide as one thatinhibits the binding of an HCV nonstructural protein to a cytoplasmicmembrane of a eukaryotic cell.
 43. The method of claim 42, wherein thepeptide is selected from SEQ ID NOS: 1-16, 18, 20-295, 297, 299-451 and454-466.
 44. An isolated peptide selected from the group consisting ofSEQ ID NOS: 1-16, 18, 20-295, 297, 299-451, and 454-466, wherein theisolated peptide causes inhibition of infection, replication, orpathogenesis of Hepatitis C Virus in vitro or in vivo when introducedinto a host cell containing the virus, and wherein the isolated peptideexhibits an IC₅₀ in the range of from about 0.0001 nM to 100 μM in an invitro assay for at least one step in infection, replication, orpathogenesis of the virus.
 45. A composition, comprising: one or moreisolated peptides of claim 44, and a carrier, diluent, excipient, orbuffer.
 46. A pharmaceutical composition, comprising: one or moreisolated peptides of claim 44, and a pharmaceutically acceptablecarrier, diluent, excipient, or buffer.
 47. A method of preventing ortreating HCV infection in a patient in need thereof, comprisingadministering to the patient an anti-HCV effective amount of one or moreisolated peptides of claim
 44. 48. Use of an isolated peptide of claim44 to prepare a medicament for the prevention or treatment of HCVinfection in a human patient in need thereof.
 49. A method of treatmentof HCV infection in a patient in need thereof, comprising administeringto the patient an anti-HCV effective amount of an isolated peptidecomprising the NS5A N-terminal amphipathic helix.
 50. The compound ofclaim 8, which is a peptide having the following amino acid sequence:SGSX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈ X₁₉X₂₀QSKL wherein X₁is W, A or F; X₂ is L, A or K; X₃ is R, A or N; X₄ is D, A or S; X₅ isV, I, D or S; X₆ is W, A or F; X₇ is D, A or S; X₈ is W, A or F; X₉ isI, E or L; X₁₀ is C, A or S; X₁₁ is T, E, A or W; X₁₂ is V, D or S; X₁₃is L, A or K; X₁₄ is T, S, A or W; X₁₅ is D, A or S; X₁₆ is F, D, or L;X₁₇ is K, A or L; X₁₈ is T, A or W; X₁₉ is W, A or F; and X₂₀ is L, A orK.